CN1230209C - Abnormal decelled bone based material and its preparation - Google Patents
Abnormal decelled bone based material and its preparation Download PDFInfo
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- CN1230209C CN1230209C CN 200310110871 CN200310110871A CN1230209C CN 1230209 C CN1230209 C CN 1230209C CN 200310110871 CN200310110871 CN 200310110871 CN 200310110871 A CN200310110871 A CN 200310110871A CN 1230209 C CN1230209 C CN 1230209C
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Abstract
The present invention relates to xenogenic cell removed bone matrix material and a preparing method thereof. The preparing method comprises: ribs or limb bones of pigs are treated physically and chemically, and cells and xenogenic protein in tissues are removed under the combined action of one of a plurality of preferred proteolytic enzymes and Triton-X100. The material maintains natural meshes and trabecular structure of bone tissues as well as ingredients and biodynamic intensity approximate to those of natural bones. The material has the advantages of low antigenicity, no obvious influence on the whole and local immunity of receptors after implantation, favorable compatibility and bone conductibility, abundant sources, convenient manufacture and low cost. The material not only can be used as filling material for bone defects, but also can be used for constructing tissue engineering bones by cell factor composition and seed cell implantation to be applied to clinical bone defect repair.
Description
Technical field
The present invention relates to that bone is damaged, bone does not connect is repaired required substitute and make up the material of bone tissue engineer,
It specifically is the xenogenesis extracellular matrix material.
Background technology
Host material as bone defect repair and bone tissue engineer can be divided into following several: nature bone and derivant thereof; Synthetic material; The complex that various materials are formed etc.Wherein, nature bone and derivant (through the bone of the whole bag of tricks processing) have near the reticulated cell slot sytem from the body bone, and The Nomenclature Composition and Structure of Complexes meets physiological requirement, and has the good cell compatibility, help the growth of seed cell, so aspect bone tissue engineer, obtained using more widely.Be divided into homogeneous allogenic bone and xenogenesis bone again by its source.By contrast, the xenogenesis bone photo is abundant to originating, cheap.But, there is the immunogenicity problem in the both.At present, generally be to reduce its antigenicity, as inorganic bones such as forging bone, deproteinization bones or remove the organic principle material such as decalcification bone, bone matrix gelatin of bone ore deposit composition fully by methods such as lyophilizing, high-temperature calcination, radiation gamma and decalcifications.Yet inorganic bone does not wherein contain organic composition, thereby loose frangible, does not possess basic mechanical intensity; The latter lacks the bone ore deposit, thereby the quality softness, does not possess basic mechanical rigidity.These deficiencies have all limited their application [Liu Jianzhong, Wang Zhen, the Chinese surgical magazine of Hu Yunyu etc. " the allograph bone joint transplantation is repaired the post-operative complication of limbs large segmental bone defect ", 2000,38 (5): 332～335] in bone tissue engineer.And there is the cost height in synthetic material, unstable properties, and mechanical strength is low, a little less than the bone-inducting active, problems such as osteogenic ability difference.Even on composition and structure, also be difficult to compare with the natural structure of osseous tissue and biomechanics and biological characteristics with material that nature bone substrate has a height bio-imitability.
Summary of the invention
First purpose of the present invention is the shortcoming that overcomes existing various xenogenesis bones, and a kind of antigen of both having eliminated is provided, and has good mechanical strength and rigidity again, and the xenogenesis that also is beneficial to cell adhesion, propagation takes off the cell extracellular matrix material.
Second purpose of the present invention provides a kind of method that described xenogenesis takes off the cell extracellular matrix material for preparing.
A kind of xenogenesis of realizing first purpose takes off the cell extracellular matrix material, and it is the Os Sus domestica of handling through physics and chemistry; The feature that is different from existing xenogenesis extracellular matrix material is that material of the present invention still removes to have taken off cell and antigenic Os Sus domestica.
Further: material of the present invention is to handle and gone to take off rib or the bones of limbs of cell and antigenic pig through physics and chemistry.
The preparation method that the xenogenesis of second purpose of realization takes off the cell extracellular matrix material is: get fresh Os Sus domestica, earlier it being carried out conventional physics and chemistry handles, comprise and remove soft tissue and bone marrow, flushing, soak in distilled water etc., crucial is that this preparation method also has following steps at least: the Os Sus domestica after 1. physics and chemistry being handled is put into H
2O
2Middle reaction is to destroy a cell and a matter albumen; 2. putting into distilled water again soaks, cleans; 3. put into methanol-chloroformic solution defat; 4. put into dehydrated alcohol and remove unnecessary chloroform; 5. put into the solution that composition comprises NaOH, Triton-X100 and proteolytic enzyme and react, to slough a cell and a matter albumen; 6. put into dehydrated alcohol and eliminate residual chloroform; 7. this bone being dried, encapsulate the back uses
60The Co radiation sterilization.Wherein, described proteolytic enzyme is Dispase enzyme or pepsin or trypsin; Described fresh Os Sus domestica mainly refers to rib or the bones of limbs of pig.
Further: the 1. used H of step
2O
2Concentration be 30%, the response time is (to destroy cell fully and a matter albumen is as the criterion) about 12 hours.Methanol, the chloroform ratio of step in 3. be about 1: 1～and 1: 4, degreasing time is 12～24 hours (being as the criterion with defat fully).NaOH content is 0.1～1.0N in the step solution 5.; Triton-X100 content is 1%～5%; The content of proteolytic enzyme is 2%～8%; Response time is 18～42 hours (being as the criterion sloughing cell fully); Obviously, for keeping the activity of enzyme, reaction temperature should be about 37 ℃.
60The exposure dose of Co is 25～30kGy.
The build size of pig is more approaching with the people, and osseous tissue is also similar, and pig is more approaching than animals such as cattle, sheep in genetic background with the people.So, adopt this xenogenesis bone of Os Sus domestica to be more suitable in material as bone tissue engineer and bone defect repair.Different with the antigenic method of existing removal, the present invention removes with the method for sloughing cell to be present in antigenic in cell and the tissue---because, be present in cell and extracellular non-collagen foreign protein has antigenicity, and mainly be present on the cell, slough a cell and a matter albumen, just removed cell and antigen naturally.Step of the present invention is simple, easy operating, and the source of material and medicine is abundant.Compared with prior art have following advantage:
1, adopt the xenogenesis extracellular matrix material of method for removing cells preparation, acellular and fragment is residual because through enzymolysis and Triton-X100 synergy, noncollagen protein is removed thoroughly, so antigenicity is extremely low, and whole body and the local immunity of implanting receptor are not had obvious influence.
2, adopt the extracellular matrix material that the cell free method of Os Sus domestica is prepared, have the mesh and the porosity of natural bone tissue, have suitable seed cell and blood vessel to grow into; Help that seed cell sticks, the three-D space structure of propagation, differentiation and phenotypic expression.And obviously be better than synthetic material aspect histocompatibility, biological degradability and the cost.
3, described material has good mechanical strength and rigidity; Have good bone conductibility and certain osteoinductive.Can be used as the damaged packing material of bone, also can make up tissue engineered bone, repairing bone defect.
4, described material is used
60The Co radiation sterilization, effectively kill virus and antibacterial are eliminated the trouble and worry of biological product clinical practice.Certainly, it also is feasible reaching other reliable sterilizing methods of this same purpose.
To further disclose described material in " specific embodiment " is made into block or Powdered.Comparatively speaking, pig rib is fit to make blocky, and its bones of limbs is fit to make pulverous.The wherein blocky cell extracellular matrix material of taking off has with fresh bone and organizes proximate biomechanical strength, rigidity and identical trabecularism, especially is fit to the damaged reparation of bones of limbs; Pulverous take off compound adhesion agent of cell bone matrix and informational molecule (as rh-BMP, VEGF etc.) after, as injectable type material repairing bone defect, bone does not connect, be characterized in little to the body wound.
Below only test and further confirm advantage of the present invention with the part of the bulk material in " specific embodiment ".
Experiment 1:, repair damaged 20 examples of rabbit radius 1.0cm with the blocky cell xenogenesis extracellular matrix material of taking off.During 8 weeks, see new bone formation; During 16 weeks, X line and histology all see bony union, and new vessels is grown into.
Experiment 2: with the blocky cell xenogenesis extracellular matrix material of taking off, compound rhBMP-2 repairs that rabbit radius 1.0cm bone is damaged, bone does not connect 15 examples respectively.The time see that new bone formation, blood flow, skeletonization amount test 1 and significantly increase 4 weeks.8 weeks were seen bony union.
Experiment 3: take off cell xenogenesis extracellular matrix material and rabbit is implanted from the body bone with blocky respectively.Implant 1～6 week of back, detect immunocyte variation in peripheral blood, the local organization, lymphproliferation response and blast transformation etc. 5 times.Both relatively do not have the statistics difference, and topographic histology is observed and seen that bone inductive effect is arranged.
Above-mentioned experiment also further shows: take off the compound rhBMP-2 of cell xenogenesis extracellular matrix material with this law made and repair that rabbit radius bone is damaged obviously to shorten healing time than traditional material, effect is remarkable, no immunological rejection.
The specific embodiment
Embodiment 1: a kind of xenogenesis takes off the preparation method of cell extracellular matrix material: getting body weight is the fresh rib of the pig of 70～100Kg, removes soft tissue and bone marrow, as required, (be the replication experiment of introducing previously, the size of this example is 1cm to make bulk
3About).Rinse well with tap water, the time is no less than 30 fens; Reuse distilled water immersion 45 minutes.After taking-up is dried, at ambient temperature, in concentration 30% H
2O
2Middle reaction 12 hours is to destroy a cell and a matter albumen.Distilled water immersion 40 minutes.After taking-up is dried, under room temperature condition, be defat 24 hours in 1: 1 the methanol chloroform solution in concentration.The reuse anhydrous alcohol solution, remove unnecessary chloroform, the time is 24 hours.After taking-up was dried, under 37 ℃ ± 1.5 ℃ condition, reaction was 20 hours in the solution that contains the NaOH of 0.8N, 2% Triton-X100,5% Dispase enzyme, to slough cell.Then, the reuse anhydrous alcohol solution is also thoroughly removed remaining chloroform, and the time is 48 hours, changes this ethanol once after the 24th hour.Take out and thoroughly dry.After the goods encapsulation, use
60Co radiation sterilization, exposure dose are 25kGy.So far, a kind of blocky xenogenesis takes off the cell extracellular matrix material and prepared and finish, and is standby.
Embodiment 2: a kind of xenogenesis takes off the preparation method of cell extracellular matrix material: getting body weight is the fresh rib of the pig of 70～100Kg, removes soft tissue and bone marrow, as required, (be the replication experiment of introducing previously, the size of this example is 1cm to make bulk
3About).Rinse well with tap water, the time is no less than 30 fens; Reuse distilled water immersion 50 minutes.After taking-up is dried, at ambient temperature, in concentration 30% H
2O
2Middle reaction 12 hours is to destroy a cell and a matter albumen.Distilled water immersion 35 minutes.After taking-up is dried, under room temperature condition, be defat 18 hours in 1: 2 the methanol chloroform solution in concentration.The reuse anhydrous alcohol solution, remove unnecessary chloroform, 32 hours time.After taking-up was dried, under 37 ℃ ± 1.5 ℃ condition, reaction was 36 hours in containing the NaOH of 0.2N, 1.5% Triton-X100,2.5% pepsic solution, to slough cell.Then, the reuse anhydrous alcohol solution is also thoroughly removed remaining chloroform, and the time is 48 hours, changes this ethanol once after the 24th hour.Take out and thoroughly dry.After the goods encapsulation, use
60Co radiation sterilization, exposure dose are 30kGy.So far, a kind of blocky xenogenesis takes off cell bone matrix timbering material and prepared and finish, and is standby.
Embodiment 3: a kind of xenogenesis takes off the preparation method of cell extracellular matrix material: getting body weight is the fresh rib of the pig of 70～100Kg, removes soft tissue and bone marrow, as required, (be the replication experiment of introducing previously, the size of this example is 1cm to make bulk
3About).Rinse well with tap water, the time is no less than 30 fens; Reuse distilled water immersion 60 minutes.After taking-up is dried, at ambient temperature, in concentration 30% H
2O
2Middle reaction 12 hours is to destroy a cell and a matter albumen.Distilled water immersion 30 minutes.After taking-up is dried, under room temperature condition, be defat 12 hours in 1: 4 the methanol chloroform solution in concentration.The reuse anhydrous alcohol solution, remove unnecessary chloroform, 48 hours time.After taking-up was dried, under 37 ℃ ± 1.5 ℃ condition, reaction was 18 hours in containing the NaOH of 1.0N, 4.5% Triton-X100,8% tryptic solution, to slough cell.Then, the reuse anhydrous alcohol solution is also thoroughly removed remaining chloroform, and the time is 48 hours, changes this ethanol once after the 24th hour.Take out and thoroughly dry.After the goods encapsulation, use
60Co radiation sterilization, exposure dose are 25kGy.So far, a kind of blocky xenogenesis takes off cell bone matrix timbering material and prepared and finish, and is standby.
Embodiment 4; A kind of xenogenesis takes off the preparation method of cell bone matrix timbering material: getting body weight is the fresh bones of limbs of the pig of 70～100Kg, removes soft tissue and bone marrow, makes the bulk about 5mm * 5mm * 5mm.Rinse well with tap water, the time is no less than 30 fens; Reuse distilled water immersion 60 minutes.After taking-up is dried, at ambient temperature, in concentration 30% H
2O
2Middle reaction 12 hours is to destroy a cell and a matter albumen.Distilled water immersion 40 minutes.After taking-up is dried, under room temperature condition, be defat 20 hours in 1: 2 the methanol chloroform solution in concentration.The reuse anhydrous alcohol solution, remove unnecessary chloroform, 36 hours time.After taking-up was dried, under 37 ℃ ± 1.5 ℃ condition, reaction was 38 hours in the solution that contains the NaOH of 0.4N, 2% Triton-X100,4% Dispase enzyme, to slough cell.Then, the reuse anhydrous alcohol solution is also thoroughly removed remaining chloroform, and the time is 48 hours, changes this ethanol once after the 24th hour.After taking-up is thoroughly dried, grind into its granular size at 0.2～1mm
3Powdered.After the goods encapsulation
60Co radiation sterilization, exposure dose are 30kGy.So far, a kind of pulverous xenogenesis takes off cell bone matrix timbering material and prepared and finish, and is standby.
Embodiment 5: a kind of xenogenesis takes off the preparation method of cell bone matrix timbering material: getting body weight is the fresh bones of limbs of the pig of 70～100Kg, removes soft tissue and bone marrow, makes the bulk about 5mm * 5mm * 5mm.Rinse well with tap water, the time is no less than 30 fens; Reuse distilled water immersion 50 minutes.After taking-up is dried, at ambient temperature, in concentration 30% H
2O
2Middle reaction 12 hours is to destroy a cell and a matter albumen.Distilled water immersion 50 minutes.After taking-up is dried, under room temperature condition, be defat 28 hours in 1: 1 the methanol chloroform solution in concentration.The reuse anhydrous alcohol solution, remove unnecessary chloroform, 32 hours time.After taking-up was dried, under 37 ℃ ± 1.5 ℃ condition, reaction was 32 hours in containing the NaOH of 0.6N, 3% Triton-X100,5% pepsic solution, to slough cell.Then, the reuse anhydrous alcohol solution is also thoroughly removed remaining chloroform, and the time is 48 hours, changes this ethanol once after the 24th hour.After taking-up is thoroughly dried, grind into its granular size at 0.2～1mm
3Powdered.After the goods encapsulation
60Co radiation sterilization, exposure dose are 25kGy.So far, a kind of pulverous xenogenesis takes off cell bone matrix timbering material and prepared and finish, and is standby.
Embodiment 6: a kind of xenogenesis takes off the preparation method of cell bone matrix timbering material: getting body weight is the fresh bones of limbs of the pig of 70～100Kg, removes soft tissue and bone marrow, makes the bulk about 5mm * 5mm * 5mm.Rinse well with tap water, the time is no less than 30 fens; Reuse distilled water immersion 60 minutes.After taking-up is dried, at ambient temperature, in concentration 30% H
2O
2Middle reaction 12 hours is to destroy a cell and a matter albumen.Distilled water immersion 30 minutes.After taking-up is dried, under room temperature condition, be defat 18 hours in 1: 3 the methanol chloroform solution in concentration.The reuse anhydrous alcohol solution, remove unnecessary chloroform, 48 hours time.After taking-up was dried, under 37 ℃ ± 1.5 ℃ condition, reaction was 20 hours in containing the NaOH of 0.8N, 5% Triton-X100,6% tryptic solution, to slough cell.Then, the reuse anhydrous alcohol solution is also thoroughly removed remaining chloroform, and the time is 48 hours, changes this ethanol once after the 24th hour.After taking-up is thoroughly dried, grind into its granular size at 0.2～1mm
3Powdered.After the goods encapsulation
60Co radiation sterilization, exposure dose are 30kGy.So far, a kind of pulverous xenogenesis takes off cell bone matrix timbering material and prepared and finish, and is standby.
From the embodiment of above-mentioned preparation method, be not difficult to find out, among the present invention about product---the summary of the invention of " xenogenesis takes off the cell extracellular matrix material " has also disclosed simultaneously, so enumerated the embodiment of this material itself no longer in addition.
Except that Os Sus domestica, can use other animal bone equally, prepare as xenogenesis bones such as Os Bovis seu Bubali, Os Caprae seu Ovis, according to method of the present invention and take off the cell extracellular matrix material, for clinical practice.
Claims (5)
1, a kind of xenogenesis takes off the cell extracellular matrix material, and it is the Os Sus domestica of handling through physics and chemistry, it is characterized in that, this material is an Os Sus domestica of having sloughed antigenic protein in a cell and the matter through egg under hydrolytic enzyme and Triton-X100 synergy.
2, a kind of xenogenesis takes off the preparation method of cell extracellular matrix material, gets fresh Os Sus domestica, earlier it is carried out conventional physics and chemistry and handles, comprise and remove soft tissue and bone marrow, flushing is soaked in distilled water, it is characterized in that this preparation method also has following steps at least: the Os Sus domestica after 1. physics and chemistry being handled is put into H
2O
2In reaction, to destroy a cell and a matter albumen, 2. putting into distilled water soaks, cleans, 3. put into methanol-chloroformic solution defat, 4. put into anhydrous alcohol solution and remove unnecessary chloroform, 5. put into the solution reaction that contains NaOH, Triton-X100 and proteolytic enzyme, to slough the albumen that a cell and a matter cause immunological rejection, 6. put into dehydrated alcohol, dissolve, eliminate residual chloroform, 7. this bone is dried, encapsulates the back and use
60The Co radiation sterilization.
3, xenogenesis according to claim 2 takes off the preparation method of cell extracellular matrix material, it is characterized in that, rib or bones of limbs that described fresh Os Sus domestica is a pig.
4, xenogenesis according to claim 2 takes off the preparation method of cell extracellular matrix material, it is characterized in that H
2O
2Concentration be 30%; Methanol, chloroform ratio are 1: 1～1: 4; NaOH content is 0.1～1.0N, and Triton-X100 content is 1%～5%, and the content of proteolytic enzyme is 2%～8%;
60The exposure dose of Co is 25～30kGy.
5, xenogenesis according to claim 2 takes off the preparation method of cell extracellular matrix material, it is characterized in that, described proteolytic enzyme is Dispase enzyme or pepsin or trypsin.
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| CN 200310110871 CN1230209C (en) | 2003-11-05 | 2003-11-05 | Abnormal decelled bone based material and its preparation |
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| CN 200310110871 CN1230209C (en) | 2003-11-05 | 2003-11-05 | Abnormal decelled bone based material and its preparation |
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| CN1230209C true CN1230209C (en) | 2005-12-07 |
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Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100496623C (en) * | 2006-06-14 | 2009-06-10 | 中国人民解放军第三军医大学第一附属医院 | Heterogenic deproteinized osseous stent material and its preparation method |
| CN101496915B (en) * | 2009-01-16 | 2012-11-21 | 中国人民解放军第三军医大学第一附属医院 | Heterogeneous dermis reticular layer stent without basement membrane and cell as well as preparation method thereof |
| CN102743793B (en) * | 2011-08-02 | 2015-05-20 | 四川大学 | Composite material, preparation method and application 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 |
| CN107456603A (en) * | 2016-06-03 | 2017-12-12 | 香港大学深圳医院 | A kind of bone holder material and preparation method for being enriched with magnesium ion |
| CN106510900A (en) * | 2016-12-08 | 2017-03-22 | 大连裕辰科技发展有限公司 | Same-kind costicartilage material for nasal part plastic filling and preparation method therefor |
| CN106730003A (en) * | 2016-12-11 | 2017-05-31 | 戴琪 | A kind of preparation method for luring bone development type bone cement |
| CN111110918B (en) * | 2019-09-26 | 2020-08-21 | 中新棠国业(苏州)医疗科技有限公司 | Preparation method of high-strength implant-grade bone material |
| CN111069149A (en) * | 2019-12-20 | 2020-04-28 | 河北鑫康辰生物技术有限公司 | Medical method for cleaning allogeneic bone |
| CN114276974A (en) * | 2021-12-24 | 2022-04-05 | 上海理工大学 | Interstitial material for encapsulating cells and preparation method and application thereof |
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