CN101172165A - Biological bone renovating material - Google Patents
Biological bone renovating material Download PDFInfo
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- CN101172165A CN101172165A CNA2007100314386A CN200710031438A CN101172165A CN 101172165 A CN101172165 A CN 101172165A CN A2007100314386 A CNA2007100314386 A CN A2007100314386A CN 200710031438 A CN200710031438 A CN 200710031438A CN 101172165 A CN101172165 A CN 101172165A
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Abstract
The invention provides biological type bone repair material, the preparation is that the repair material takes an animal bone as the raw material and is formed by degreasing, extracting cell, removing antihelion from multiple directions, fixing epoxy, processing 1N sodium hydroxide, inducing active decoration and the irradiation of a Gamma ray, and disinfecting and killing virus and other processes. The biological type bone repair material of the invention effectively removes immunogenicity, also keeps bone matrix including multiple active components, has bone induced activity, is adjacent to human bodies on organic composition and inorganic composition and multi-dimension structure, and can well induce the regeneration of bone tissue. In addition, the application is safer, and the histocompatibility and the mechanical property are good.
Description
Technical field
The present invention relates to a kind of bone renovating material, be used for the reparation damaged, belong to and implant the class medical apparatus and instruments, be specially biological bone renovating material bone.
Background technology
The caused bone of surgical operation or pathological changes is damaged to be one of surgical common disease, and the treatment that bone is damaged need be used a large amount of bone renovating materials, promptly usually said artificial bone.The bone renovating material that is studied at present and uses mainly contains two big classes: a class is the bone renovating material of being made by synthetic material, mainly contains hydroxyapatite, tricalcium phosphate, polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), polycaprolactam (PA
6) etc.Outside inorganic skeleton composition in above-described artificial bone hydroxyl-removal apatite, tricalcium phosphate and the osseous tissue was similar, the composition of all the other high polymers and bone had no common ground, was as a kind of shape stent applications.Hydroxyapatite and tricalcium phosphate, no matter sinter molding or the above high polymer are made the binding agent bonding forming, and its mechanical strength is all undesirable.Low fire ceramic not only property is crisp, and too hard, and host bone tissue is difficult to grow into, and therefore difficult and osseous tissue combines together.So how present hydroxyapatite and tricalcium phosphate are used as bone filler with powdery, granule or mastic, application is restricted; Second class is to be the bone renovating material of raw material with the nature bone.What be employed the earliest is animal bone, uses after organic matter is removed in 600～800 ℃ of roastings.The effect of roasting is an organic matter of removing strong immune immunogenicity, simultaneously killing pathogenic bacteria virus.But the sclerotin of roasting is very crisp, the mechanical strength variation, and behind the burning-off bone matrix, induce the ability of osseous tissue growth, can only among a small circle, use as inserts.At present by with the most use be the corpse bone, use after generally only soaking sterilization treatment as freezing and simple ethanol, ray sterilizing or glutaraldehyde, immune rejection often takes place and influence therapeutic effect.The source of main is corpse bone scarcely is donations voluntarily, even having is voluntary donations on a small quantity, by various countries' relevant laws, the organ of donations is that can not to be used for the profit be the transaction of purpose, in the market, the problem that has an ethics, the human rights, law as the corpse bone of commodity.Along with perfecting of law legal system, the source of corpse bone will be very limited.The research of much attempting to replace with animal bone the corpse bone is arranged in recent years, and some is just just used according to the simple deep cooling and the alcohol-pickled processing of corpse bone; Some carries out a little complicated processing such as defat, hydrofluoric acid treatment, glutaraldehyde immersion etc., all fails to design the antigen measure of going accordingly at the stronger immune immunogenicity of bone matrix.Therefore, is the artificial bone repair materials of raw material at present with the animal bone, exist the immune immunogenicity of can not ignore mostly and the osseous tissue growth healing that is difficult to induce the host, the problem that repairing effect is not good enough, thereby do not accepted by the user.Although many researcheres claim that all the animal bone of oneself handling is heterogeneous bone free of antibody or does not have antigens heterogeneity bone, but it is because not enough to immunogenic essence understanding, fail targetedly deblocking or destroy epitope (claiming antigenic determinant again), take for simple deep cooling or glutaraldehyde and soak and just can effectively remove antigen.Actually this is not so, according to immunoreactive molecular biology research and immunochemical result of study are thought, the immunogenicity of xenogenesis osseous tissue is to cause that by some active group or special conformation that is in special sensitive position in the bone matrix these specific activity groups or special conformation are called as epitope or antigenic determinant.Its antigenicity to be effectively removed and the structure of epitope must be managed to seal or destroy targetedly, and can not lean on simply freezing, defat, glutaraldehyde to handle, can not be only by taking off cell because acellular connective tissue, even the collagen of purifying all has certain antigenicity.
Summary of the invention
The objective of the invention is deficiency at the above artificial bone existence, providing a kind of is raw material with the animal bone, it is similar to nature bone to form (containing organic and inorganic composition) and structure (comprising that micropore runs through the multidimensional structure of structure), no antigen, virus-free, histocompatibility good, can induce the biological bone renovating material of receptor bone tissue growth healing.
The present invention is achieved in that biological bone renovating material is is raw material with the animal bone, is made through following making step:
A, choose suitable animal bone: comprise compact bone and spongy bone etc.;
B, pretreatment: carry out disinfection, removal of impurity, machine cuts molding etc.;
C, defat: with the fatty impurity of organic solvent extracting animal bone inside;
D, take off cell: remove wherein cell with surfactant or protease;
E, the fixing processing: the ossein in the material is carried out crosslinked fixing as cross linking fixative with epoxide;
F, the multi-faceted antigen that removes: plurality of reagents is destroyed or the blocking antigen epi-position;
G, Protein virus are killed: kill the Protein virus that may exist;
H, induced activity are modified: can adhere to somatomedin and cell activity component, and introduce biological bone surface;
I, packing;
J, gamma-radiation radiation sterilization;
K, through the check after get product.
In the above B step, the used disinfectant of described sterilization mainly is general broad-spectrum disinfectant, as benzalkonium bromide, hibitane, Hydrazoic acid,sodium salt etc.;
In the above C step, the organic solvent of the fatty impurity of extracting animal bone inside can be acetate esters, chloroform, carbon tetrachloride, ether, acetone, dehydrated alcohol etc.;
In the above D step, take off cell and be with the surfactant elution method and in the presence of collagen is protectant enzymatic isolation method, both use separately or both should be used for realizing simultaneously; Surfactant can be ethylenediaminetetraacetic acid, benzyl fluosulfonic acid, Triton X-100 (Tritonx100), Tris (Tris), and the used enzyme of enzymatic isolation method can be trypsin and pepsin.
In the above E step, described epoxy fixedly is meant and the ossein in the material is carried out crosslinked fixing with epoxide as cross linking fixative.Here epoxide can be the carbochain epoxide
, R=H, CH in the formula
3(CH
2)
n-or
, n=0,1,2 ... n; It also can be carbon oxygen chain epoxide
, R=C in the formula
nH
2n+1-,
, R '=H, CH
3-(C
2H
5)
n-, n=0,1,2 ... n, x=0,1,2 ... x.Epoxy cross-linking is fixing than the fixed benefit of traditional glutaraldehyde cross-linking to be: because epoxy radicals is more active, crosslinkedly carry out very fully, and easily control the degree of cross linking and correlated performance by the consumption of cross-linking agent, cross-linking products is more stable, not degraded easily, under the driving that cell proliferation, tissue growth need, go out the collaborative collagenase effect of kallikrein by emiocytosis, just with its degraded and absorbed, be passive degraded state, can make degraded and tissue growth synchronous, and its catabolite non-residual toxicity, histocompatibility is good.
In the above F step, the described multi-faceted antigen that removes is meant with plurality of reagents destruction or blocking antigen epi-position.Comprise with nucleopilic reagent combining its sealing with causing antigenic specific activity group, and cause antigenic special conformation with the change of strong hydrogen bonding reagent, wherein the conformation of tropocollagen molecule is mainly formed by hydrogen bond and causes; Specific activity group described here mainly is-OH
*,-NH
2 *,-SH
*Deng.Described nucleopilic reagent can be an acyl ammonia
, R=C in the formula
nH
2n+1-, n=0,1,2 ... n; Amide
, R=C in the formula
nH
2n+1-, n=0,1,2 ... n; Anhydride
, R=C in the formula
nH
2n+1-, n=1～10; Epoxide
, R=C in the formula
nH
2n+1-,
, n=0,1～10 etc., described strong hydrogen bonding reagent mainly is guanidine compound.
In the above F step, it is to kill the Protein virus that may exist that described Protein virus is killed, and can be no less than 1 hour with 1N soaking with sodium hydroxide bone material.
In the above G step, described induced activity modification is meant and can adheres to somatomedin and cell activity component, complex as some specific polypeptide or glycosaminoglycans is introduced product surface (containing the micropore inner surface), make to adhere to somatomedin and the seed cell that enrichment body self-regeneration mechanism discharges after implanting, induce the osseous tissue growth efficiently; Also can implant at the external BMP that adheres to earlier, BMP is a bone morphogenetic protein here again.Specific polypeptide described here is the polypeptides matter that a class can non-specific adhesion somatomedin, and one of polypeptide is served as reasons and contained the polypeptide that 16 lysines (K) and glycine (G), arginine (R), aspartic acid (D), serine (S), proline (P), cysteine (C) aggregate into: K (16) GRGDSPC; The glycosaminoglycans complex can be the complex of hyaluronic acid, chondroitin sulfate, keratan sulfate, heparin, heparan etc.The method of introducing can be a coupling agent coupling mode, also can chemisorbed.Coupling agent mainly be some easily with-NH
2,-OH, the difunctional material that the active hydrogen of-SH group reacts for example can be two acid diamides, two acyl dichloros, diacid intramolecular anhydride, di-epoxide, carbodiimides etc.
Among above-mentioned main preparation process C, D, E, F, G, the H, all used high infiltration technology simultaneously, in high permeable reactive device, carried out.So-called high infiltration technology is exactly the high permeable reactive device of a sonic oscillation and vacuum pulse interlock, can effectively gas in the bone material micropore and granule foreign be discharged by sonic oscillation and vacuum pulse, reagent corresponding can be worked the infiltration into microporous depths, guarantee inside and outside effect fully.
After the packing of product that makes,, can effectively kill other known animal derived viruses except that Protein virus with the cobalt-60 gamma-radiation irradiation sterilization that is not less than 25KGy.Like this, it is similar to nature bone just to make composition and structure, aseptic, virus-free, can not cause immune rejection, and good biocompatibility can be induced the novel artificial bone material of osteanagenesis.
Biological bone renovating material of the present invention can be made kinds of artificial bone (being that bone substitutes body) as required, comprise artificial rib, phalanges, breastbone, skull etc., also can be made into bone fixation means such as bone screw, hone lamella, intramedullary needle, spinal fusion device, also can be made into filling material of bone such as skeletal grain, bone piece, bone bar, be applied in the orthopaedics therapy.
The advantage of biological bone renovating material of the present invention is: be raw material with the full bone of animal directly, by epoxy fix, the multi-faceted advanced technologies such as antigen and induced activity modification of removing handle, and used high osmosis process, made effective removal immunogenicity, kept the sclerotin base that contains numerous active components again, novel artificial bone material with bone-inducting active, all more approaching on organic and inorganic composition and multidimensional structure with the human body bone, can induce osteanagenesis better.And use NaOH simultaneously and kill Protein virus and use gamma-radiation irradiation and kill known viruse, make use safer.Organic matter (deproteinization) animal bone is gone in contrast, and artificial bone of the present invention is all even better on histocompatibility, mechanical property.Antigen is handled and epoxy is fixing handles without removing specially in contrast, and the Protein virus that goes out is handled, only do that simply alcohol-pickled, defat, glutaraldehyde are fixed, the animal bone of gamma-radiation sterilization, biological bone renovating material of the present invention histocompatibility and safety, go aspect immunogenicity, the osteoinductive even better.
Description of drawings
Fig. 1 makes the cross-sectional view of artificial bone rib for biological bone renovating material of the present invention;
Fig. 2 makes the bone screw sketch map for biological bone renovating material of the present invention;
Fig. 3 makes the sketch map of spinal fusion device for biological bone renovating material of the present invention
The specific embodiment
The present invention will be described in detail below in conjunction with specific embodiment.
Embodiment 1
Biological bone renovating material is to be raw material with the animal bone, is made through following making step:
A, choose animal bone;
B, pretreatment: with 0.1% benzalkonium bromide sterilization 30 minutes, clean the back specific purpose tool,, eliminate muscular tissue and periosteum, wash clean as xyster etc.;
C, defat: wherein fatty with the acetas lixiviate in high permeable reactive device, clean then;
D, take off cell: in high permeable reactive device, take off cell, clean then with surfactant;
E, the fixing processing: in high permeable reactive device, use epoxidation reagent
Fix processing, clean R=C in the formula then
nH
2n+1-,
, n=0,1,2 ... n; F, the multi-faceted antigen that removes: in high permeable reactive device, use the acyl chlorides nucleopilic reagent
Sealing causes antigenic active group, cleans R=C in the formula then
nH
2n+1-, n=0,1,2 ... n;
G, Protein virus are killed: be no less than 60 minutes with the 1N soaking with sodium hydroxide in high permeable reactive device, clean;
H, induced activity modify: in high permeable reactive device, polypeptide K (16) GRGDSPC of non-specific adhesion somatomedin be coupled in the goods with coupling agent carbodiimides (R-N=C=N-R '), and clean;
I, packing: pack with the sterile saline liquid storage hygrometric state of going bail for;
J, gamma-radiation radiation sterilization: with the sterilization of cobalt 60 gamma-radiations, gamma ray dose is not less than 25Kgy;
K, through the check after get product.
Embodiment 2
F, the multi-faceted antigen that removes are used the amide nucleopilic reagent in high permeable reactive device
Sealing causes antigenic active group, cleans R=C in the formula then
nH
2n+1-, n=0,1,2 ... n;
H, induced activity are modified, and with coupling agent diacid intramolecular anhydride polypeptide K (16) GRGDSPC are coupled in the goods in high permeable reactive device;
All the other steps are similar, and are no longer burdensome.
Embodiment 3
B, pretreatment:, clean the back with eliminating muscular tissue and periosteum, wash clean with 0.1% peracetic acid disinfectant 30 minutes;
E, the fixing processing: in high permeable reactive device, use epoxidation reagent
Fix processing, clean R=C in the formula then
nH
2n+1-,
R '=H, CH
3-(C
2H
5)
n-, n=0,1,2 ... n, x=0,1,2 ... x.
F, the multi-faceted antigen that removes: in high permeable reactive device, use epoxide
With the antigenic active group of guanidine compound sealing causing, clean R=C in the formula then
nH
2n+1-,
, n=0,1～10;
H, induced activity are modified: with coupling agent two acyl dichloros polypeptide K (16) GRGDSPC is coupled in the goods in high permeable reactive device;
All the other steps are similar, and are no longer burdensome.
In the preparation method of biological bone renovating material of the present invention, specialized designs use the multi-faceted antigen technology of removing that multiple active reagent sealed and destroyed epitope from many aspects, in addition, also designed with the above gamma-radiation radiation of 25KGy and killed animal originality virus, and increased the active surface activity modification step of osteogenic induction.Consider also that in addition bone matrix mainly exists in the micropore of bone, for the reagent that makes each step reaction can go deep in the micropore and the bone matrix effect, specialized designs one cover sonic oscillation add the high permeable reactive device of vacuum pulse, guarantee that reaction reagent energy infiltration into microporous depths and bone matrix react, and guarantee that effect is more abundant more effective.Make biological bone renovating material of the present invention aspect histocompatibility, osteogenic induction activity, the practicality all than existing be that the goods of raw material are superior with the animal bone.
Biological bone renovating material of the present invention can be made kinds of artificial bone (claiming bone to substitute body again) as required, comprises artificial rib, phalanges, breastbone, skull etc.; Also can be made into bone fixation means such as hone lamella, intramedullary needle, bone screw, spinal fusion device, also can be made into skeletal grain, bone bar, bone piece, filling material of bone etc.
Claims (10)
1. biological bone renovating material, it is characterized in that: with the animal bone is raw material, comprises that being prepared as follows step makes:
A, choose suitable animal bone;
B, pretreatment: carry out disinfection, the removal of impurity, machine cuts molding;
C, defat: with the fatty impurity of organic solvent extracting animal bone inside;
D, take off cell: remove wherein cell with surfactant or protease;
E, the fixing processing: the ossein in the material is carried out crosslinked fixing with cross linking fixative;
F, the multi-faceted antigen that removes: use plurality of reagents and destroy or the blocking antigen epi-position;
G, Protein virus are killed: kill the Protein virus that may exist;
H, induced activity are modified: can adhere to somatomedin and cell activity component, and introduce biological bone surface;
I, packing;
J, gamma-radiation radiation sterilization: kill the animal derived pathogenic bacteria or the virus that may exist;
K, through the check after get product.
2. biological bone renovating material according to claim 1 is characterized in that: described C, D, E, F, G, H preparation process are carried out in high permeable reactive device.
3. biological bone renovating material according to claim 1 is characterized in that: the pretreated sterilization of B step is soaked with broad-spectrum disinfectant solutions such as benzalkonium bromide, hibitane, peracetic acid, Hydrazoic acid,sodium salt and is realized.
4. biological bone renovating material according to claim 1 is characterized in that: the organic solvent of C step defat is acetate esters, chloroform, carbon tetrachloride, ether, acetone, dehydrated alcohol.
5. biological bone renovating material according to claim 1 is characterized in that: the cell that takes off in the D step is with surfactant elution method and the enzymatic isolation method in the presence of the collagen protective agent, and both use separately or both should be used for realizing simultaneously; Surfactant is ethylenediaminetetraacetic acid, benzyl fluosulfonic acid, Triton X-100, Tris, and the used enzyme of enzymatic isolation method is trypsin and pepsin.
6. biological bone renovating material according to claim 1 is characterized in that: the used epoxide of fixedly processing in the E step has:
Perhaps
, R=C in the formula
nH
2n+1-,
, R '=H, CH
3-(C
2H
5)
n-, n=0,1,2 ... n, x=0,1,2 ... x.
7. biological bone renovating material according to claim 1 is characterized in that being: it is nucleopilic reagent and strong hydrogen bonding reagent that multi-faceted in the F step removes antigenic reagent, and used nucleopilic reagent has acyl ammonia, amide, anhydride or epoxide; Strong hydrogen bonding reagent is guanidine compound.
8. biological bone renovating material according to claim 1 is characterized in that: it is that specific polypeptide or glycosaminoglycans complex with coupling agent can adhere to somatomedin and cell is coupled in the biological bone that induced activity in the H step is modified; Described specific polypeptide is the polypeptides matter that a class can non-specific adhesion somatomedin, and one of this polypeptides matter is for containing the polypeptide of 16 lysines (K) and glycine (G), arginine (R), aspartic acid (D), serine (S), proline (P), cysteine (C): K (16) GRGDSPC; Described glycosaminoglycans complex is the complex of hyaluronic acid, chondroitin sulfate, keratan sulfate, heparin, heparan; Described coupling agent be comprise two acid diamides, diacid intramolecular anhydride, two acyl dichloros, di-epoxide easily and the bifunctional reagent that reacts of the active hydrogen in polypeptide and the ossein.
9. biological bone renovating material according to claim 1 is characterized in that: it is to soak biological bone material with 1N NaOH to be no less than 60 minutes that the Protein virus of G step is killed step.
10. biological bone renovating material according to claim 1, it is characterized in that: it is used to make Biotype artificial rib, phalanges, breastbone, skull, bone screw, hone lamella, intramedullary needle, spinal fusion device, and the bone filling is applied in the orthopaedics therapy with bone reparation goods such as skeletal grain, bone piece, bone bars.
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|---|---|---|---|
| CNA2007100314386A CN101172165A (en) | 2007-11-16 | 2007-11-16 | Biological bone renovating material |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100314386A CN101172165A (en) | 2007-11-16 | 2007-11-16 | Biological bone renovating material |
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Family
ID=39421051
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| CN104174068B (en) * | 2013-05-22 | 2016-02-17 | 烟台正海生物科技股份有限公司 | A kind of biotype cartilage repair material and preparation method thereof |
| CN103638557B (en) * | 2013-10-24 | 2015-05-27 | 陕西佰傲再生医学有限公司 | Antigen-removing biological bone and preparation method thereof |
| CN103638557A (en) * | 2013-10-24 | 2014-03-19 | 陕西佰傲再生医学有限公司 | Antigen-removing biological bone and preparation method thereof |
| CN104788692A (en) * | 2015-04-30 | 2015-07-22 | 上海欣吉特生物科技有限公司 | Inactivated collagen material and preparation method thereof |
| CN109908402A (en) * | 2017-12-12 | 2019-06-21 | 冠昊生物科技股份有限公司 | A kind of cancellous bone collagen plugs and preparation method thereof |
| CN110193093A (en) * | 2018-02-24 | 2019-09-03 | 上海优先生物医学工程有限公司 | A kind of new soft tissue filling renovation material and its preparation method and application |
| CN110960339A (en) * | 2019-11-26 | 2020-04-07 | 广州迈普再生医学科技股份有限公司 | Skull repairing body and skull repairing system |
| TWI751674B (en) * | 2020-09-01 | 2022-01-01 | 元進莊企業股份有限公司 | The use of poultry bone sintered materials for bone filling materials |
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