CN2817772Y - Tissue engineering bone graft - Google Patents
Tissue engineering bone graft Download PDFInfo
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- CN2817772Y CN2817772Y CNU2005200570196U CN200520057019U CN2817772Y CN 2817772 Y CN2817772 Y CN 2817772Y CN U2005200570196 U CNU2005200570196 U CN U2005200570196U CN 200520057019 U CN200520057019 U CN 200520057019U CN 2817772 Y CN2817772 Y CN 2817772Y
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- bone graft
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Abstract
The utility model belongs to the technical field of building artificial organs with a tissue engineering method in the biomedical engineering, which particularly relates to a tissue engineering bone graft. The tissue engineering bone graft of the utility model comprises a carrier supporting frame and a seed cell, wherein the seed cell is attached on the carrier supporting frame to form a composite body which has bone tissue three-dimensional structure and bioactivity. The carrier supporting frame is modified PLGA which is provided with a large aperture and high porosity and is treated through deacidification, and is loaded with cytokine bone morphogenetic protein, namely BMP. The seed cell is a bone marrow stem cell. The tissue engineering bone graft of the utility model is a functional tissue engineering bone graft which can be used for making and repairing a large section of bone defect.
Description
Technical field
This utility model belongs in the biomedical engineering with Method of Tissue Engineering and makes up the artificial organ technical field, specifically relates to a kind of organizational project bone graft.
Background technology
In recent years, development along with tissue engineering technique, the report that research and development have reparation, keep or improve the biological substitution thing of damaged tissue function increases gradually, wherein bone tissue engineer is studied a big focus especially, has formed the comparatively perfect theory and technology route about seed cell, cell carrier support and tissue construction at present gradually.But undeniable is that these adjusted range bone tissue engineers finally still have certain distance in Clinical Application.Therefore, be necessary to develop and a kind ofly can be applied to clinical novel organizational project bone graft product rapidly.
The basic way of organizational project artificial tissue is that the tissue that will in vivo obtain is dispersed into single cell suspension with Mechanical Method or enzyme digestion, hatches cultivation then under the conditions in vitro of simulated in vivo environment, makes cell survival, growth and amplification.Then will be to three-dimensional stent material with certain space structure at the certain density cell seeding of having of In vitro culture, further cultivate, by the mutual adhesion between the cell, growth, breeding, secretory cell epimatrix, form tissue and organ with certain 26S Proteasome Structure and Function.The cell of In vitro culture should have most of function of cells in vivo.Osteoblast as In vitro culture has high alkaline phosphatase activity, secretion Bone Gla protein, synthetic justacrine I, II Collagen Type VI etc.There are some researches show that when existing, osteoblast can form bone trabecula in culture bottle in calcification condition (calcium ion, sodium, vitamin C, dexamethasone etc.).Therefore, utilize Method of Tissue Engineering that osteoblast is inoculated on certain timbering material, and the cytokine of adding some promote osteogenesis and bone neural blood vesselization cultivates external, it is fully feasible obtaining an osseous tissue.
Maximum problem is the selection of timbering material in the bone tissue engineer research at present.The required timbering material of bone tissue engineer should have following characteristics: have excellent biological compatibility and biological degradability, catabolite in vivo is harmless; 2. have certain osteoinductive and bone conductibility, be beneficial to cell attaching and propagation; 3. have certain intensity, maintenance self shape also can be to external force resistance in body; 4. be easy to mouldingly, can be processed into different shape and size as required; 5. the high osmosis that has load maximum cell; 6. support the surface chemical property and the micro structure of Oesteoblast growth and function differentiation; 7. can be with other biological bioactive molecule such as bone morphogenetic protein (BMP) thus compound, controlled release the growth of seed cell is regulated and control; 8. easy sterilization property.The bone tissue engineering stent material source can be divided into inorganic material and organic material two big classes at present.Inorganic material mainly is meant bioceramic class material.This class material mainly is made up of calcium, P elements, and is similar with main inorganic composition in the human body, so have excellent biological compatibility, biological degradability and bone conductibility.It is extremely difficult that but the greatest drawback of this class material is to degrade, and influenced the generation and the reconstruction of area of new bone.Organic material can be divided into natural and artificial organic material.Natural organic comprises collagen, chitosan, fibrin gel etc., and the general character of this class material is a good biocompatibility, is beneficial to cell and attaches, breeds, breaks up, but not enough as the bone tissue engineering scaffold mechanical strength, degradation time is difficult to control.
Bone morphogenetic protein is that ((Bone morphogenetic protein, BMP)) research skeletal growth factor early, the effect of its induced osteogenesis is confirmed by repeatedly experiment.It is the unique local growth factor that can induce osseous tissue to form separately, and under certain condition, can induce undifferentiated mesenchymal cell is cell transformation to bone, promotes the growing multiplication of osteocyte.Think that at present BMP promotes in the bone tissue engineer that ossification is most important a kind of.But because the content of BMP in osseous tissue few (about 1mg/kg wet bone), and spread very soon in vivo, decomposed by protease easily, thereby can not bring into play the effect that continues to irritate with induced osteogenesis in the part, its induced activity is difficult to be fully played.
Although organizational project bone graft development aspect has obtained huge progress, portioned product has entered clinical experimental stage, but these products are imperfection still, there is defective in all many-sides of method maturation, lead time and product cost, especially the reparation of large segmental bone defect clinically still do not had positive effect.
The utility model content
The purpose of this utility model is to provide a kind of organizational project bone graft.This organizational project bone graft after being implanted into human body, do not have rejection, within a certain period of time fully degraded and absorbed, induced osteogenesis effect obviously, can repair large segmental bone defect, lower cost.
A kind of organizational project bone graft described in the utility model, comprise carrier bracket, seed cell, described carrier bracket is the PLGA with large aperture and high porosity and the processing of process disacidify of modification, and load has cytokine bone morphogenetic protein, i.e. BMP on it; Described seed cell be bone marrow stroma stem cell (bone marrow stem cells, BMSCs).
Described seed cell is to take from same bone marrow, and In vitro culture is the bone marrow stroma stem cell in the 3rd generation through separating, increase also, and its cell density is 1 * 10
6-1 * 10
7Individual/ml.
The aperture of described PLGA carrier bracket is 150～200um, and the aperture rate is 85～95%.
The carrier bracket raw material that this utility model adopts is poly-milk alcohol acid (polylactic-glycol acid, PLGA), it is the derivant of artificial organic material PLA, possess advantages such as excellent biological compatibility, catabolite avirulence, easy processing, certain intensity, organizational project bone graft described in the utility model can be widely used.
The cytokine that this utility model adopts is bone morphogenetic protein (BMP), the regeneration that it not only can accelerated bone, but also play a part to quicken the timbering material degraded.
This utility model has carried out optimization process to PLGA timbering material three dimensional structure, can bring into play the biological efficiency of BMP to greatest extent.Employing is a raw material with the PLGA with large aperture and high porosity and the processing of process disacidify of modification, and the aperture of preferred PLGA carrier bracket is 150～200um, and the aperture rate is 85～95%, and can be processed as all size arbitrarily according to requirement of experiment.
Organizational project bone graft described in the utility model can be used for preparing the functional organization through engineering approaches bone graft of repairing large segmental bone defect.This purposes is established by the test of animal bone defect repair.
Description of drawings
Fig. 1 is the structural representation of organizational project bone graft of the present invention, is the sketch map of partly cutting open;
Fig. 2 be among Fig. 1 A-A to cutaway view.
The specific embodiment
Now this utility model and implementation result are described further in conjunction with experiment.
As shown in Figure 1, a kind of organizational project bone graft described in the utility model comprises carrier bracket 1, seed cell 2, and described carrier bracket 1 is the PLGA with large aperture and high porosity and the processing of process disacidify of modification, load has cytokine bone morphogenetic protein 3, i.e. BMP on it; Described seed cell 2 is a bone marrow stroma stem cell.
Fig. 1 is the microcosmic sketch map, and its ratio is not represented the ratio of actual product.
Described seed cell 2 is to take from same bone marrow, and In vitro culture is the bone marrow stroma stem cell in the 3rd generation through separating, increase also, and its cell density is 1 * 10
6-1 * 10
7Individual/ml.
The aperture of described PLGA carrier bracket 1 is 150～200um, and the aperture rate is 85～95%.
The construction method of organizational project bone graft described in the utility model may further comprise the steps:
A. the PLGA with large aperture and high porosity and the processing of process disacidify with modification is a raw material, adds cytokine bone morphogenetic protein, i.e. BMP in the preparation process of organizational project bone graft carrier bracket;
B. will take from same bone marrow, through the bone marrow stroma stem cell digestion that separates, amplification and In vitro culture were the 3rd generation, centrifugal, with 1 * 10
6-1 * 10
7/ ml concentration is inoculated on the PLGA carrier bracket;
C. the PLGA carrier bracket of compound cell is put into incubator and cultivated 3-6 hour, add the DMEM conditioned medium, put back to and continue in the incubator to cultivate 3-5 days, promptly obtain the organizational project bone graft.
In the above-mentioned construction method, described seed cell is that bone marrow stroma stem cell is to prepare through the following steps:
(1) obtains spongy bone bone marrow,, obtain bone marrow stroma stem cell with full bone marrow culture method flush away erythrocyte progressively in changing the liquid process;
(2) with 0.25% trypsinization, be passaged to the 3rd generation bone marrow stroma stem cell;
(3) reach the 3rd generation cell begin to change liquid with the DMEM conditioned medium, be carried out to the osteocyte directional induction, cultivate standby after 3-5 days.
Described DMEM conditioned medium comprises 15% serum, 50 μ g/ml ascorbic acid, 10
-8Mol/L dexamethasone, 10mmol/L sodium.
In the above-mentioned construction method, described PLGA support is to prepare through the following steps:
A, be raw material with the PLGA with large aperture and high porosity of modification, the cytokine bone morphogenetic protein that adds the shape of claying into power is BMP, and repaiies the type of being cut into;
B, cleaned by ultrasonic vibration;
C, surperficial disacidify processing;
D, 37 ℃ of epoxyethane fumigation sterilizations are soaked;
The DMEM complete medium that e, adding contain 15% serum soaked 2-3 days;
F, adding 10% Poly-L-Lysine Solution soaked 1-2 days;
15-25 ℃ of drying under g, the uviol lamp preserved standby.
The test of animal bone defect repair:
Get New Zealand's large ear rabbit, 2 monthly ages, body weight 1.5～2.0kg, male and female are not limit, available from Nanfang Medical Univ (former No.1 Military Medical Univ.) animal center.The organizational project bone graft damaged in the long 15mm standard bone of radius stage casing, right side preparation respectively under the aseptic condition, that implantation embodiment three is constructed is successively closed otch, postoperative animal normal activity.Matched group is respectively blank PLGA group, PLGA+BMSCs group.Put to death animal in 4,8,12 weeks, do following the detection: (1) reaches x-ray observation substantially: observe animal activity, gait, wound healing and defect repair situation, and bone defective region area of new bone is carried out optical density value relatively.(2) histological observation: get two radius stage casings bone specimen, 10% paraformaldehyde is fixed, decalcification, routine paraffin wax embedded section, HE dyeing, om observation area of new bone growing state.(3) bone defective region area of new bone is tested with carry out biomechanics with the position normal bone, done compressive strain and three point bending test.
The result:
Gross examination of skeletal muscle: visible tissue engineering bone graft group Os Leporis seu Oryctolagi is damaged when drawing materials repairs fully, and outward appearance is normal.
Phase contrast microscope is observed: as shown in Figure 2, the PLGA support of BMSCs and load BMP meets to be cultivated after 3 days, and visible cell and material attach good, and secrete a large amount of cellular matrixs, and the iuntercellular gap is unclear.
Histological observation: as shown in Figure 3, visible PLGA scaffold degradation is the mesh sample, and chondrocyte and the formation of a large amount of cartilage matrix.Existing a large amount of area of new bone formed when HE dyeing showed for 4 weeks, and each time period skeletonization all obviously is better than matched group.
X-ray observation: shown in Fig. 4 (a), the 4th all X lines show that the bone defective region has obvious new bone growth, and the interface fogs between implanting tissue engineering bone graft and the place notes bone; Shown in Fig. 4 (b), the seriality callus appears in the 8th all bone defective regions, and density obviously increases; Shown in Fig. 4 (c), the damaged reparation substantially fully of the 12nd week back bone, and it is logical again pulp cavity to occur.
Optical density detects: show organizational project bone graft composition bone amount maximum of the present invention.
Biomechanics detects: show load, bending stress significant difference between each group.
Claims (1)
1, a kind of organizational project bone graft, comprise carrier bracket (1) and seed cell (2), seed cell (2) is attached on the carrier bracket (1), formation has osseous tissue three dimensional structure and bioactive complex, it is characterized in that: described carrier bracket (1) is the PLGA with large aperture and high porosity and the processing of process disacidify of modification, load has cytokine bone morphogenetic protein (3), i.e. BMP on it; Described seed cell (2) is a bone marrow stroma stem cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNU2005200570196U CN2817772Y (en) | 2005-04-14 | 2005-04-14 | Tissue engineering bone graft |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNU2005200570196U CN2817772Y (en) | 2005-04-14 | 2005-04-14 | Tissue engineering bone graft |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN2817772Y true CN2817772Y (en) | 2006-09-20 |
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|---|---|---|---|
| CNU2005200570196U Expired - Fee Related CN2817772Y (en) | 2005-04-14 | 2005-04-14 | Tissue engineering bone graft |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1846793B (en) * | 2005-04-14 | 2011-08-03 | 南方医院 | Tissue engineering bone and its construction and application |
| CN101439204B (en) * | 2008-12-29 | 2012-09-05 | 暨南大学 | Tissue engineered bone-cartilage complex tissue implant and preparation method thereof |
-
2005
- 2005-04-14 CN CNU2005200570196U patent/CN2817772Y/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1846793B (en) * | 2005-04-14 | 2011-08-03 | 南方医院 | Tissue engineering bone and its construction and application |
| CN101439204B (en) * | 2008-12-29 | 2012-09-05 | 暨南大学 | Tissue engineered bone-cartilage complex tissue implant and preparation method thereof |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20060920 Termination date: 20140414 |