CN1200740C - Method for preparing artificial bone - Google Patents
Method for preparing artificial bone Download PDFInfo
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- CN1200740C CN1200740C CN 03112269 CN03112269A CN1200740C CN 1200740 C CN1200740 C CN 1200740C CN 03112269 CN03112269 CN 03112269 CN 03112269 A CN03112269 A CN 03112269A CN 1200740 C CN1200740 C CN 1200740C
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Abstract
The present invention relates to a method for preparing an artificial bone, which belongs to the technical field of a biomedical material. The technical problem to be solved is to provide a method for preparing an artificial bone. An artificial bone can promote vascularization, improve blood circulation and imitate the synergistic action of each factor in the osteogenetic process in a body so as to realize osteogenesis. The method has the technical scheme that the method for preparing an artificial bone comprises the working procedures: preparing a bracket, compounding active factors for osteogenesis, sterilizing and separately packing. The method for preparing an artificial bone also comprises the working procedure of compounding vascular endothelial growth factors The bracket not only compounds bone morphogenetic protein factors, transforming growth factors, etc., but also compounds the vascular endothelial growth factors and solves the problem of blood vessel formation and blood supply in the osteogenetic process of the artificial bone, the vascular endothelial growth factors can promote the cell proliferation, differentiation and vascularization of blood vessel endothelium, convey nutritive substance, osteogenetic factors, etc. required by injure restoring to fracture positions and simultaneously carry metabolites away from the fracture positions so that the osteogenetic and blood vessel forming capacity is greatly enhanced.
Description
Technical field
The present invention relates to a kind of manufacture method of medical bionical bone, belong to technical field of biomedical materials.
Background technology
Subject doctors' such as bone is damaged, bone does not connect is puzzlement orthopaedics, oral cavity always, brain is an outer difficult problem, the expert is devoted to seek ideal bone-grafting material always both at home and abroad.For a long time, taking from body bone bone grafting is a kind of method of standard, limited, especially particularly outstanding to this problem of child but this method is got the bone amount.And the quality of bone bank bone is very unstable, and the ratio of refracture is higher, and has the probability of pathophoresis.In recent years, the various countries expert has developed substitution material and the preparation technology of multiple replacement from the body bone in succession, " reconstituted bone xenograft " disclosed as Chinese patent 94244566.X, 97108538.2 disclose " lump type defatted antigen-removing heterogenic bone grafting material and preparation method thereof ", 99119851.4 disclose " a kind of novel bone renovating material ", 01108617.3 discloses " biologically cmposite artificial bone and preparation technology thereof ".In above-mentioned patent and the research of other numerous artificial bones, a lot of research worker adopt the porous crack macromolecular material of synthetic or pass through animal skeletons such as chemically treated people's bone, Os Bovis seu Bubali, Os Sus domestica as tissue engineering bracket material, compound skeletal growth factor based on bone morphogenetic protein (BMP) on these materials is to promote osteanagenesis and reparation.This technology has obtained certain progress at present, but in the technology of above-mentioned compound skeletal growth factor, does not all relate to for vascularization and blood supply behind the artificial bone-grafting material implant into body, and this is a very big shortcoming.At first, the growth of spur amount of union of fracture place or bone-grafting material filling place that studies show that is directly proportional with partial vascularization amount and blood supply amount, if vascularization and blood supply are seldom or do not have, then the skeletonization amount is very little even can not skeletonization; Secondly, the reparation of fracture is the pathological process of a complexity, synthetic and the mineralising that relates to propagation, differentiation and the extracellular matrix of various kinds of cell, along with the development of Protocols in Molecular Biology, have now found that at fracture to exist multiple somatomedin that they are collaborative mutually in fracture healing process, influence each other, mutually promote, it is not enough only adding the BMP factor, can not finish skeletonization truly.
Summary of the invention
Technical problem to be solved by this invention provides a kind of preparation method of bionical bone, and this bionical bone can promote vascularization, improves blood circulation, the multiple-factor synergism in the parody in the osteogenetic process, thus realize skeletonization well.
The scheme that solves the problems of the technologies described above is: a kind of preparation method of bionical bone, it comprises prepares one of support, the bone morphogenetic protein that is combined into the bone active factor, transforming growth factor or two kinds, sterilization, packing operation, its difference part is, it also comprises composite vascular endothelial cell growth factor (ECGF) operation, and concrete steps are:
A. vascular endothelial cell growth factor is dissolved in the medical solvent, addition is 0.001mg/ml-10mg/ml;
B. support is immersed above-mentioned solvent soaking after drying.
The preparation method of above-mentioned bionical bone, in the described composite vascular endothelial cell growth factor (ECGF) operation, said support is soak at room temperature in medical solvent, soak time is 10 minutes-24 hours, evacuation 0.01Mpa-0.10Mpa then, lyophilizing, said medical solvent are water or medical buffer, heparin solution, NaCl solution.
The preparation method of above-mentioned bionical bone, described medical buffer is a phosphate buffered saline(PBS), said sterilization process adopts ethylene oxide sterilizing.
The preparation method of above-mentioned bionical bone, described preparation support operation is:
A. choose timbering material: choose fresh Os Bovis seu Bubali, Os Sus domestica, people's bone or other animal bone, cut the spongy bone of epiphysis end or vertebral body, be processed into required form;
B. physics and chemical treatment: broken, scavenger cell, defat, removal foreign body, deproteinization are removed antigenicity;
C. cleaning, lyophilizing, packing, sterilization are preserved standby;
More than preparation support operation also can be undertaken by other prior art, or adopts the support of synthetic.
The preparation method of above-mentioned bionical bone, one of the described bone morphogenetic protein that is combined into the bone active factor, transforming growth factor or two kinds of operations are:
A. prepare bone morphogenetic protein solution: it is in the urea liquid of 2-10M that bone morphogenetic protein is dissolved in guanidine hydrochloride solution or the concentration that concentration is 4-7M, and addition is the 0.01-3.0mg/ml of guanidine hydrochloride solution or urea liquid;
B. prepare transforming growth factor solution: transforming growth factor is added in the acetum that concentration is 10mM-1M, and addition is 0.01-3.0mg/ml, soaks 0.5-1 hour, stirs;
C. compound: get that two kinds of solution that above-mentioned A, B step make mix or above-mentioned two kinds of solution in any one, will prop up and be placed in it, soaked 0.5-24 hour, be evacuated to 0.01Mpa-0.10Mpa again, with 1 ℃ of-20 ℃ of distill water dialysis 24-96 hour lyophilizing.
Support can be combined into the bone active factor earlier, again the composite vascular endothelial cell growth factor (ECGF); Also can be with support elder generation composite vascular endothelial cell growth factor (ECGF), compound again other osteogenic activity factor, or vascular endothelial cell growth factor, other osteogenic activity factor is simultaneously compound.
The preparation method of above-mentioned bionical bone also can with support and mesenchymal stem cells MSCs, osteoblast, fibroblast, fibroblast-like cells, chondroblast or to carry the above-mentioned cell of transgenic fragment compound with known technology.
The preparation method of above-mentioned bionical bone, the physical treatment step of described preparation support operation be,
A. flushing: with the pulse of 20-50 ℃ of high pressure clean water, stirring flushing;
B. dry: centrifuge dripping;
C. freezing-as to heat up: be refrigerated to-20 ℃～-80 ℃ and take out immediately, drop into 0.1 mol, pH value and be in 7.2 the buffer and placed 1-24 hour, be warming up to room temperature, this process can repeat 1-5 time, thereby makes the cell breakage of green bone, scavenger cell, promotion skeletonization.
The chemical treatment step of described preparation support operation is defat, removal foreign body, deproteinization removal antigenicity, and bone piece centrifugal speed is 100～3000 rev/mins, and the time is 5～30 minutes.
The preparation method of above-mentioned bionical bone, described being combined in the bone active factor operation, also comprise preparation collagen and composite collagen: type i collagen lyophilizing grinding is microgranular, adding concentration is in the 10mM-1M acetum, and addition is 0.1-3.0mg/ml, together soaks 0.5-1 hour with transforming growth factor, stir, to prop up again and be placed in it, leave standstill 0.5-24 hour, be evacuated to the 0.01Mpa-0.10Mpa lyophilizing then.
The preparation method of above-mentioned bionical bone, in the described composite steps that is combined into bone active factor operation, said to get above-mentioned two kinds of solution be by (1-4): (4-1) mixed, sonic oscillation stirs; For improving support intensity, in the chemical treatment operation of preparation support, described deproteinization is removed before the antigenicity step, the bone piece is put into formaldehyde or the formaldehyde/glutaraldehyde mixed liquor that concentration expressed in percentage by volume is 1%-10% handled 1-24 hour.
Vascular endothelial cell growth factor (VEGF) is the multifunctional cytokine of a kind of specific action in vascular endothelial cell, is alkalescence, meets heat and meets sour stablizing, and has high conservative.This factor pair angiogenesis plays an important role, participate in the overall process of union of fracture, especially fracture back 72 hours to 3 weeks are active stages of fracture revascularization, vascular endothelial cell growth factor (VEGF) can impel vascular endothelial cell division growth, growth, transfer strongly and specifically, increases vascular permeability and promotes neovascularity to generate.Vascular endothelial cell growth factor (VEGF) can also directly act on osteoblast, increases it and moves and differentiation function.Vascular endothelial cell growth factor (VEGF) and bone morphogenetic protein (BMP
s), have synergism between the transforming growth factor osteogenic factors such as (TGF-β), skeletonization and promote angiopoietic ability to improve greatly.Therefore vascular endothelial cell growth factor (VEGF) is compounded in the above-mentioned artificial bone-grafting material and will promotes vascularization, improve blood circulation, realize skeletonization better.
Compared with prior art, the present invention has following advantage:
In technical scheme of the present invention, bionical bone holder material is also compound vascular endothelial cell growth factor except the factors such as compound bone morphogenetic protein, transforming growth factor, this has just solved the problem of bionical bone osteogenetic process medium vessels formation and blood supply, vascular endothelial cell growth factor can promote vascular endothelial cell proliferation, differentiation and vascularization, nutrient substance that injury repairing is required and osteogenic factor etc. are transported to fracture site, while, from fracture site, skeletonization and one-tenth blood vessel ability improved greatly the metabolite band.Vascular endothelial cell growth factor influence each other between them, and biological effect can be worked in coordination with mutually as a member in the multiple somatomedin network simultaneously.In preparation process, also can adopt the temperature difference New Process for Treatment of pulsation pressure flush, freezing-intensification, improve the processing quality of timbering material greatly.
The specific embodiment
Embodiment 1
(1). the preparation support
A. choose timbering material: choose the fresh adult Os Bovis seu Bubali, cutting epiphysis end spongy bone is required form;
B. physical treatment:
A. flushing: stir flushing with 30 ℃ of high pressure clean water pulsation;
B. dry: with 500 rev/mins speed centrifuge dripping;
C. freezing-as to heat up: put into refrigerator-freezer, be refrigerated to-80 ℃ and take out immediately, drop into 0.1 mol, PH and be in 7.2 the phosphate buffer and soaked 20 hours, rise to room temperature, freezing again, soak, carry out 2-4 time repeatedly;
C. chemical treatment:
A. defat, removal foreign body: under 30 ℃, with 1: 1 chloroform/methanol solution soaking defat 12 hours, with bone piece centrifugal 10 minutes with 500 rev/mins speed, remove the foreign body in the bone piece, the sodium hydroxide solution of putting into 20% concentration again soaked 18 hours, the foreign body in cleaning, the centrifugal removing bone piece;
B. deproteinization, remove antigenicity
The bone piece in being 30% hydrogen peroxide solution, 30 ℃, concentration expressed in percentage by volume was soaked the foreign body in the centrifugal again removing bone piece 12 hours;
C. repeating step a, b;
D. carry out ultrasonic waves for cleaning, distilled water flushing with known method, lyophilizing, packing, sterilization, preservation are standby;
(2). compound bone morphogenetic protein, collagen
A. prepare the bone morphogenetic protein guanidine hydrochloride solution
The bone morphogenetic protein that extracts is dissolved in the guanidine hydrochloride solution, and addition is the 1.0mg/ml of guanidine hydrochloride solution, and the concentration of guanidine hydrochloride is 5M;
B. prepare collagen and transforming factor solution
The type i collagen lyophilizing grinding of extracting is microgranular, adds in the acetum, addition is the 0.5mg/ml of acetum, the transforming growth factor that extracts is also added in the above-mentioned acetum, and addition is 0.5mg/ml, soaks 1 hour, stir, the concentration of acetic acid is 1M;
C. composite bionic bone holder material
Above-mentioned two kinds of solution are mixed, and sonic oscillation stirs, and both ratios are 1: 4, and bionical bone holder material is placed in it, leave standstill 12 hours, are evacuated to 0.09Mpa then, with 4 ℃ of distill water dialysis lyophilizing after 48 hours;
(3). the composite vascular endothelial cell growth factor (ECGF)
The vascular endothelial cell growth factor that extracts is dissolved in the PBS solution, addition is 5mg/ml, the bionical bone holder material of compound bone morphogenetic protein, collagen, transforming growth factor is put in this PBS solution again, left standstill 12 hours, temperature is 4 ℃, be evacuated to the 0.09Mpa lyophilizing, ethylene oxide sterilizing, packing is standby.
Embodiment 2
(1). the preparation of support: before the b. deproteinization in the chemical treatment operation of embodiment 1 preparation support is removed the antigenicity step, timbering material dropped in 5% formaldehyde and the glutaraldehyde mixed solution (mixed proportion 1: 1) immersion 12 hours, other step is with embodiment 1.
(2). compound bone morphogenetic protein and transforming growth factor
A. the bone morphogenetic protein that extracts is dissolved in the guanidine hydrochloride solution, addition is the 2.0mg/ml of guanidine hydrochloride solution, and the concentration of guanidine hydrochloride is 6M; The transforming growth factor that extracts is added in the acetum, and addition is the 0.7mg/ml of acetum, fully soaks 1 hour, stirs, and the concentration of acetic acid is 1M.
B. above-mentioned two kinds of solution are mixed, sonic oscillation stirs, and both ratios are 1: 1; Drop into bionical bone holder material, left standstill 20 hours, be evacuated to 0.09Mpa again, with 4 ℃ of distill water dialysis lyophilizing after 72 hours;
(3). the composite vascular endothelial cell growth factor (ECGF)
The vascular endothelial cell growth factor that extracts is dissolved in the PBS solution, and addition is 5mg/ml; The bionical bone holder material of compound bone morphogenetic protein, transforming growth factor is put in the above-mentioned PBS solution, left standstill 20 hours, temperature is 4 ℃, evacuation, lyophilizing; Ethylene oxide sterilizing, packing is standby.
Skeletal growth factor of the present invention can be that genetic engineering is produced, and also can extract from various animal tissues such as cortical bone, platelet, Medulla Bovis seu Bubali, people; The factors such as bone morphogenetic protein can be albumen or the heterodimer of its excalation thing or the associating gene expression products of polymer or several genes of modified, as BMP
2-BMP
7, BMP
7-TGF-β, BMP
7-VEGF etc., can be on support antibiotic, antitubercular agent, antineoplastic chemotherapy medicine such as compound gentamycin.
Claims (9)
1. the preparation method of a bionical bone, it comprises prepares one of support, the bone morphogenetic protein that is combined into the bone active factor, transforming growth factor or two kinds, sterilization, packing operation, it is characterized in that, it also comprises composite vascular endothelial cell growth factor (ECGF) operation, and the concrete steps of this operation are:
A. vascular endothelial cell growth factor is dissolved in the medical solvent, addition is 0.001mg/ml-10mg/ml;
B. support is gone into above-mentioned solvent soaking after drying.
2. according to the preparation method of the described bionical bone of claim 1, it is characterized in that, in described composite vascular endothelial cell growth factor (ECGF) operation, said support is soak at room temperature in medical solvent, soak time is 10 minutes-24 hours, evacuation 0.01Mpa-0.10Mpa then, lyophilizing, said medical solvent is water or medical buffer, heparin solution, NaCl solution.
3. according to the preparation method of the described bionical bone of claim 2, it is characterized in that described medical buffer is a phosphate buffered saline(PBS), said sterilization process adopts ethylene oxide sterilizing.
4. according to the preparation method of claim 1 or 2 described bionical bones, it is characterized in that described preparation support operation is:
A. choose timbering material: choose fresh Os Bovis seu Bubali, Os Sus domestica, people's bone or other animal bone, cut the spongy bone of epiphysis end or vertebral body, be processed into required form;
B. physics and chemical treatment: broken, scavenger cell, defat, removal foreign body, deproteinization are removed antigenicity;
C. cleaning, lyophilizing, packing, sterilization.
5. according to the preparation method of claim 1,2 or 3 described bionical bones, it is characterized in that one of the described bone morphogenetic protein that is combined into the bone active factor, transforming growth factor or two kinds of operations are:
A. prepare bone morphogenetic protein solution: it is in the urea liquid of 2-10M that bone morphogenetic protein is dissolved in guanidine hydrochloride solution or the concentration that concentration is 4-7M, and addition is the 0.01-3.0mg/ml of guanidine hydrochloride solution or urea liquid;
B. prepare transforming growth factor solution: transforming growth factor is added in the acetum that concentration is 10mM-1M, and addition is 0.01-3.0mg/ml, soaks 0.5-1 hour, stirs;
C. compound: get that two kinds of solution that above-mentioned A, B step make mix or above-mentioned two kinds of solution in any one, will prop up and be placed in it, left standstill 0.5-24 hour, be evacuated to 0.01Mpa-0.10Mpa again, with 1 ℃ of-20 ℃ of distill water dialysis 24-96 hour lyophilizing.
6. according to the preparation method of claim 1 or 2 described bionical bones, it is characterized in that, with support and mesenchymal stem cells MSCs, osteoblast, fibroblast, fibroblast-like cells, chondroblast or to carry the above-mentioned cell of transgenic fragment compound.
7. according to the preparation method of the described bionical bone of claim 4, it is characterized in that:
The physical treatment step of described preparation support operation is a. flushing: with the pulse of 20-50 ℃ of high pressure clean water, stirring flushing; B. dry: centrifuge dripping; C. freezing-as to heat up: as to be refrigerated to-20 ℃～-80 ℃ and to take out immediately, drop into 0.1 mol, pH value and be in 7.2 the buffer and placed 1-24 hour, be warming up to room temperature, carry out 1-5 time;
The chemical treatment step of described preparation support operation is defat, removal foreign body, deproteinization removal antigenicity, and the centrifugal speed of bone piece is 100～3000 rev/mins, and the time is 5～30 minutes.
8. according to the preparation method of the described bionical bone of claim 4, it is characterized in that, be combined in the bone active factor operation described, also comprise preparation collagen and composite collagen: type i collagen lyophilizing grinding is microgranular, and adding concentration is in the 10mM-1M acetum, addition is 0.1-3.0mg/ml, together soaked 0.5-1 hour with transforming growth factor, stir, will prop up again and be placed in it, left standstill 0.5-24 hour, and be evacuated to the 0.01Mpa-0.10Mpa lyophilizing then.
9. according to the preparation method of the described bionical bone of claim 5, it is characterized in that in the described composite steps that is combined into bone active factor operation, said to get above-mentioned two kinds of solution be by (1-4): (4-1) mixed, sonic oscillation stirs; In the chemical treatment operation of preparation support, described deproteinization is removed before the antigenicity step, the bone piece is put into formaldehyde or the formaldehyde/glutaraldehyde mixed liquor that concentration expressed in percentage by volume is 1%-10% soaked 1-24 hour.
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| CN 03112269 CN1200740C (en) | 2003-05-28 | 2003-05-28 | Method for preparing artificial bone |
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| CN 03112269 CN1200740C (en) | 2003-05-28 | 2003-05-28 | Method for preparing artificial bone |
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| CN1200740C true CN1200740C (en) | 2005-05-11 |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100457187C (en) * | 2006-11-10 | 2009-02-04 | 中国人民解放军第二军医大学 | VEGF slowly releasing injection microsphere support and its prepn and use |
| CN102697548B (en) * | 2012-06-21 | 2014-07-23 | 闫宏伟 | Method for preparing novel individual degradable artificial intraosseous stent |
| CN112999422A (en) * | 2021-03-23 | 2021-06-22 | 潍坊奥精医学研究有限公司 | Preparation method of bone repair material |
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