CN101168072A - Artificial bone and its making method - Google Patents
Artificial bone and its making method Download PDFInfo
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- CN101168072A CN101168072A CNA2006101047903A CN200610104790A CN101168072A CN 101168072 A CN101168072 A CN 101168072A CN A2006101047903 A CNA2006101047903 A CN A2006101047903A CN 200610104790 A CN200610104790 A CN 200610104790A CN 101168072 A CN101168072 A CN 101168072A
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Abstract
Disclosed are an artificial bone and a manufacturing method of the artificial bone. Cell membranes form a prototype of the artificial bone, the cell membranes are cultivated in culture fluid of inducing action, and then the cell membranes are mutually engaged, finally the artificial bone is achieved. The composition of the artificial bone comprises bone marrow stromal cells after being induced and stromas which are secreted by the cells, histology represents a chondroid tissue, the coloration of safranine -O shows a positive reaction, and when the artificial bone is implanted into a hypoderm of a naked mouse, a mature bone tissue can be formed. The invention solves technical problems that alternate materials of bones of the background technique produce chronic inflammation reaction or rejection owning to the existing of a support, the long term remaining of foreign matters, or the degrading of products. According to the sizes and shapes of damaged bones, a reproduced bone tissue without supports are a complete biological tissue of the functions of safe and reliable usage and being capable of completely replacing damaged bones.
Description
Technical field
The present invention relates to a kind of bone alternate material, be specifically related to a kind of complete artificial bone and manufacture method thereof that constitutes by the substrate of cell and emiocytosis.
Background technology
Contemporary medical science is damaged for the bone that a variety of causes such as wound, bone tumor pathological changes cause, bone does not connect etc., does not still have effective treatment means, and main armrest art is fixed, connected, or uses bone-grafting material and from body bone, bone xenograft.Because healing stage is very long, easily cause various complication, wherein manyly do not disable because there being effective treatment means.
Organizational project is the new subject that development in recent years is got up.Its most basic thinking is: at in-vitro separation, cultured cell, with cell seeding to three-dimensional rack with certain space structure, by the mutual adhesion between the cell, growth and breeding, secretory cell epimatrix, form tissue or organ with certain 26S Proteasome Structure and Function.Because timbering material can constantly be degraded along with neoblastic formation, finally absorbed fully, the tissue of producing again and identical from the body normal structure, thus reproduce out biological tissue or organ.
Chinese invention patent, number of patent application is 02145459, relates to a kind of tissue-engineered bone and preparation method thereof.The concrete form that the bone that can repair as required is damaged carries out moulding to engineered bone holder material.The skin of gained tissue-engineered bone is the cortical bone framework, and the middle part has the spongy bone succedaneum of bone marrow interstital stem cell to substitute spongy bone and bone marrow composition in the normal bone structure by inoculation.Its tissue-engineered bone has the cortical bone similar to normal bone tissues, spongy bone and cell component.In the clinical practice, the doctor can measure the excision scope in advance according to patient's CT sheet, and by damaged partial C T reconstruct data engineered bone holder material is carried out moulding, thereby realize individualized treatment.
Chinese invention patent, application number 00132082 is a bio-derived bone support of Os Sus domestica or allograph bone being made basic removal antigenicity and cell component, reservation basic framework, being used for inoculating cell becomes tissue engineered bone.The preparation method of the disclosed tissue engineered bone of United States Patent (USP) (6,228,117) is to prepare the polyactide macromolecular material earlier, and inoculation has the cell of skeletonization potential then, becomes bone alternate material, promotes the damaged reparation of bone.
The common ground of above-mentioned technology is: all have support in its bone alternate material, promptly support all will implant with bone alternate material.Bone tissue engineering stent material commonly used has bioceramic, synthesized polymer material, natural macromolecular material.Bioceramic has hydroxyapatite, tricalcium phosphate, hydroxyapatite-tricalcium phosphate biphase ceramics, bone pottery etc.; Synthesized polymer material has polylactic acid, polyglycolic acid, polycaprolactone, poly butyric ester etc.; Natural macromolecular material has collagen protein, chitin, demineralized bone etc.Because the material of these supports itself is different from osseous tissue, therefore all there is deficiency in various degree.For example: bioceramices such as hydroxyapatite can not be degraded, and will retain for a long time as foreign body after implanting; The catabolite of macromolecular material etc. can cause chronic inflammatory reaction; There is certain rejection in big right macromolecular material such as collagen protein, demineralized bone owing to derive from allosome or heteroplasm.
Summary of the invention
The object of the present invention is to provide a kind of artificial bone and manufacture method thereof, it has solved in the background technology in the bone alternate material because of there being support, or causes foreign body to retain for a long time, or catabolite causes chronic inflammatory reaction, or has the technical problem of rejection.
The technology of the present invention solution is as follows:
A kind of artificial bone, its formation comprise the bone marrow stroma stem cell after inducing and the substrate of these emiocytosises; The histology shows as chondroid tissue; Sarranine-O dyeing is positive; Implant in the nude mice subcutaneous tissue and can form sophisticated osseous tissue.
A kind of artificial bone, it forms the artificial bone blank with cell patch, obtains through fusion again; Constitute the bone marrow stroma stem cell comprise after inducing and the substrate of these emiocytosises; The histology shows as chondroid tissue; Sarranine-O dyeing is positive; Implant in the nude mice subcutaneous tissue and can form sophisticated osseous tissue.
A kind of method of making above-mentioned artificial bone, implementation step comprises:
(1) form the artificial bone blank: cell patch is wrapped in forming mould surface, or directly folding, the blank of formation artificial bone;
(2) diaphragm merges: the artificial bone blank is cultivated 2-10 week in having the culture fluid of inducing action, merge mutually to cell patch, and directly must artificial bone; Or must artificial bone behind the removal shaping dies.
The above-mentioned acquisition of stating cell patch may further comprise the steps:
(1) obtains bone marrow stroma stem cell: get bone marrow, must be present in the bone marrow stroma stem cell in the bone marrow, carry out anticoagulant;
(2) cultivation of bone marrow stroma stem cell amplification: culture fluid, the bone marrow after the anticoagulant that will have inducing action are inserted in the culture vessel, with the culture fluid with inducing action bone marrow stroma stem cell are cultivated amplification;
(3) obtain cell patch: the continuous culture amplification, the bone marrow stroma stem cell profile after inducing disappears, and has the mineralising tuberosity to occur, and promptly gets cell patch.
Above-mentioned diaphragm merges and cultivates amplification all is to carry out under the condition that biological cell keeps surviving.
Above-mentioned culture fluid with inducing action comprises that volume fraction is the serum of 5-15%, the vitamin C of 5-100 mcg/ml, 10 with the basal liquid of DMEM or DMEM/F12 preparation
-9-10
-6The dexamethasone of M, the sodium of 5-20mM; The glutamine that also can comprise 0.2-0.4g/L.
Above-mentioned bone marrow can adopt body bone marrow, the allosome bone marrow that also can adopt tissue matching to select.
Above-mentioned shaping dies can be made of tubing, sheet material or mesh material, and the shape of described shaping dies is corresponding with damaged shape.
Above-mentioned shaping dies can adopt metal material, macromolecular material or bioceramic material etc.
The present invention has the following advantages:
(1) only needs a spot of body or allosome bone marrow, can obtain enough cell quantities and be used for transplanting.
(2) not having support in the osseous tissue of producing again, is complete biological tissue, has not both had the worry that causes chronic inflammatory disease, does not also have the melancholy of the rejection of causing.
(3) artificial bone has significantly reduced the healing, the reparation phase that implant at In vitro culture.
(4) can be shaped according to shape, the size of defective bone.Or direct forming is extremely corresponding with damaged shape, or is shaped by a plurality of amalgamations.
(5) safe in utilization, reliable, can substitute the function of defective bone fully.
Description of drawings
Fig. 1 is in contact with one another, connects observation figure under the inverted microscope in blocks for cell of the present invention;
The cell patch figure of Fig. 2 for obtaining among the present invention;
Fig. 3 is artificial bone blank figure of the present invention;
The tubular artificial bone figure that Fig. 4 can be used for transplanting for the present invention;
Fig. 5 shows as the figure of chondroid tissue, sarranine-O stained positive for the histology of the artificial bone of the present invention;
Fig. 6 is for having calcium deposition, von-kossa stained positive figure among the present invention manually;
Fig. 7 implants the ripe osseous tissue figure that forms in the nude mice subcutaneous tissue for the artificial bone of the present invention.
The specific embodiment
The artificial bone of the present invention, rice is cultivated amplification with the culture fluid with inducing action to bone marrow stroma stem cell and is obtained cell patch, forms the artificial bone blank with cell patch, obtains through fusion again.Substrate by the bone marrow stroma stem cell after inducing and these emiocytosises constitutes, and the histology shows as chondroid tissue, and sarranine-O dyeing is positive.Implant in the nude mice subcutaneous tissue, can form sophisticated osseous tissue.
The manufacture method of the artificial bone of the present invention is as follows:
1. obtain bone marrow stroma stem cell: get bone marrow, must be present in the bone marrow stroma stem cell in the bone marrow, carry out anticoagulant.Bone marrow can be body bone marrow, also can be the allosome bone marrow of selecting by tissue matching.Anticoagulant is adopted in anticoagulant, and preferred heparin also can adopt sodium citrate.
2. the cultivation of bone marrow stroma stem cell amplification: culture fluid, the bone marrow after the anticoagulant that will have inducing action are inserted in the culture vessel, keep at biological cell by culture fluid bone marrow stroma stem cell being cultivated amplification under the condition of survival with inducing action.Culture fluid with inducing action, its basal liquid is prepared by DMEM or DMEM/F12, and generally contain at least: volume fraction is the serum of 5-15%, the vitamin C, 10 of 5-100 mcg/ml
-9-10
-6M dexamethasone and 5-20mM sodium can contain the glutamine of 0.2-0.4g/L in addition.34-39 ℃ of the condition temperature of cultivating amplification all can, be advisable with 37 ± 0.5 ℃.Generally getting normal condition is: 37 ℃, saturated humidity, 5%CO
2Condition.
3. acquisition cell patch: the continuous culture amplification, the bone marrow stroma stem cell profile of observation after induce disappears and has the mineralising tuberosity to occur under the inverted microscope, promptly gets cell patch.
4. form the artificial bone blank: cell patch is wrapped in forming mould surface, or directly folding, the blank of formation artificial bone.Shaping dies is used for tubular bone more, and its shape is corresponding with damaged shape.The artificial bone blank that directly is folded to form generally is used for the non-tubular shape bone, and its shape that can directly fold is corresponding with damaged shape, also can be pieced together corresponding with damaged shape by two or more.Shaping dies is made of tubing, or surrounds tubulose by sheet material or mesh material.Shaping dies is the best to adopt Web materials, and it can make the culture fluid effect with inducing action to the cell patch inboard, is more conducive to the cultivation of cell patch.The material of shaping dies is generally metal, macromolecular material or bioceramic etc.As: rustless steel, nylon etc.
5. diaphragm merges: under standard conditions, the blank of artificial bone is continued cultivation 2-10 week in having the culture fluid of inducing action, merge mutually to cell patch, promptly get artificial bone.Or the removal shaping dies gets artificial bone.The diaphragm time of fusion is generally 2-10 week, can select according to the size and the growing state of artificial bone blank.
Experiment embodiment 1:
(1) extracts 3ml bone marrow from crista iliaca under the aseptic condition, use anticoagulant heparin.
(2) change bone marrow over to 75cm
2In the culture bottle, bone marrow stroma stem cell is cultivated amplification with culture fluid with inducing action.Culture fluid with inducing action is DMEM, wherein contains 10% serum, 50 μ g/ml vitamin Cs, 10
-8M dexamethasone, 10mM sodium.
(3) continuous culture amplification, the culture fluid that replacing in every 3-4 days once has inducing action.Observe with inverted microscope, the bone marrow stroma stem cell after inducing is in contact with one another, connects in flakes, profile disappears and have the mineralising tuberosity to occur.The X100 inverted microscope is observed figure down, sees Fig. 1.With scraper it is lifted from the culture bottle bottom, obtain cell patch, referring to Fig. 2.
(4) cell patch is wrapped in the forming mould surface of the tubulose stainless (steel) wire of diameter 4mm, long 3cm, forms the blank of tubular artificial bone, see Fig. 3.
(5) tubular artificial bone blank is continued to cultivate for 6 weeks with the culture fluid with inducing action, cell patch is merged mutually, remove shaping dies then, become the tubular artificial bone that can be used for transplanting, see Fig. 4.
Experimental result:
With tubular artificial bone 10% formalin fixed, histological examination is carried out in gradient alcohol dehydration, waxdip, embedding, section, dyeing, the showed cell diaphragm merges mutually, artificial bone is a chondroid tissue, and sarranine-O dyeing is positive, and X400 microscopically observation figure sees Fig. 5.Wherein do not contain exogenous timbering material.Calcium deposition is arranged, and von-kossa dyeing is positive, and the X100 microscopically is observed figure, sees Fig. 6.
Experiment embodiment 2:
When the osseous tissue of implanting when needs is not tubulose, the cell patch that forms in the experiment embodiment 1 directly is folded into the box-shaped of about 10 * 10 * 2mm, forms the blank of artificial bone.External continuation cultivated for 4 weeks, formed the box-shaped artificial bone.
Experimental result: aseptic condition is implanted down in the nude mice subcutaneous tissue, forms sophisticated osseous tissue after 2 months, and the X100 microscopically is observed figure, sees Fig. 7.
Claims (9)
1. artificial bone, its formation comprise the bone marrow stroma stem cell after inducing and the substrate of these emiocytosises; The histology shows as chondroid tissue; Sarranine-O dyeing is positive; Implant in the nude mice subcutaneous tissue and can form sophisticated osseous tissue.
2. artificial bone, it forms the artificial bone blank with cell patch, obtains through fusion again; Constitute the bone marrow stroma stem cell comprise after inducing and the substrate of these emiocytosises; The histology shows as chondroid tissue; Sarranine-O dyeing is positive; Implant in the nude mice subcutaneous tissue and can form sophisticated osseous tissue.
3. method of making claim 1 or 2 described artificial bones, implementation step comprises:
(1) form the artificial bone blank: cell patch is wrapped in forming mould surface, or directly folding, the blank of formation artificial bone;
(2) diaphragm merges: the artificial bone blank is cultivated 2-10 week in having the culture fluid of inducing action, merge mutually to cell patch, and directly must artificial bone; Or must artificial bone behind the removal shaping dies.
4. according to the manufacture method of the described artificial bone of claim 3, it is characterized in that the obtaining to lead of described cell patch may further comprise the steps:
(1) obtains bone marrow stroma stem cell: get bone marrow, must be present in the bone marrow stroma stem cell in the bone marrow, carry out anticoagulant;
(2) cultivation of bone marrow stroma stem cell amplification: culture fluid, the bone marrow after the anticoagulant that will have inducing action are inserted in the culture vessel, with the culture fluid with inducing action bone marrow stroma stem cell are cultivated amplification;
(3) obtain cell patch: the continuous culture amplification, the bone marrow stroma stem cell profile after inducing disappears, and has the mineralising tuberosity to occur, and promptly gets cell patch.
5. according to the manufacture method of claim 3 or 4 described artificial bones, it is characterized in that: described diaphragm merges, cultivates amplification and all carries out under the condition that biological cell keeps surviving.
6. according to the manufacture method of the described artificial bone of claim 5, it is characterized in that described culture fluid with inducing action comprises the basal liquid of DMEM or DMEM/F12 preparation, volume fraction is the serum of 5-15%, the vitamin C of 5-100 mcg/ml, 10
-9-10
-6The dexamethasone of M, the sodium of 5-20mM; Or also comprise the glutamine of 0.2-0.4g/L.
7. according to the manufacture method of the described artificial bone of claim 6, it is characterized in that: described bone marrow is body bone marrow, or joins the allosome bone marrow that type is selected by carefully knitting.
8. according to the manufacture method of the described artificial bone of claim 7, it is characterized in that: described shaping dies is made of tubing, sheet material or mesh material, and the shape of described shaping dies is corresponding with damaged shape.
9. the manufacture method of described artificial bone according to Claim 8, it is characterized in that: described shaping dies is metal material, macromolecular material or bioceramic material.
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| CNA2006101047903A CN101168072A (en) | 2006-10-23 | 2006-10-23 | Artificial bone and its making method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102526806A (en) * | 2012-01-20 | 2012-07-04 | 陕西博鸿生物科技有限公司 | Tissue engineering cartilage and preparation method thereof |
| CN105854085A (en) * | 2016-04-25 | 2016-08-17 | 上海国睿生命科技有限公司 | Method for constructing tissue engineered cartilages in vivo |
-
2006
- 2006-10-23 CN CNA2006101047903A patent/CN101168072A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102526806A (en) * | 2012-01-20 | 2012-07-04 | 陕西博鸿生物科技有限公司 | Tissue engineering cartilage and preparation method thereof |
| CN102526806B (en) * | 2012-01-20 | 2013-12-18 | 陕西博鸿生物科技有限公司 | Tissue engineering cartilage and preparation method thereof |
| CN105854085A (en) * | 2016-04-25 | 2016-08-17 | 上海国睿生命科技有限公司 | Method for constructing tissue engineered cartilages in vivo |
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Open date: 20080430 |