CN1733318A - Construction method of sclerotomal cell idiosyncratic transcription factor - 2 gene decorated tissue engineered bone - Google Patents
Construction method of sclerotomal cell idiosyncratic transcription factor - 2 gene decorated tissue engineered bone Download PDFInfo
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Abstract
Disclosed is a construction method of sclerotomal cell idiosyncratic transcription factor - 2 gene decorated tissue engineered bone through employing a natural biologically derived material (acellular bone matrix) and seed cells (osteoblast specific transcription factor-2 gene modified mesen chymal stem cells), carrying out extracorporal dynamic culture and restoration for large segment long bone defection. The method can realize the clinic cicatrisation of bone defections.
Description
Technical field
The present invention is the construction method of the tissue-engineered bone of osteoblast idiosyncratic transcription factor-2 genetic modification, belongs to the bone tissue engineer technical field.
Background technology
Because the bone that tumor resection, wound, osteopathia and bone misgrowth are caused is damaged, simple rely on to repair voluntarily be difficult to healing, so bone grafting operation is the effective measures of treatment large segmental bone defect.Become the tissue transplantation's technology that is only second to blood transfusion at U.S.'s bone grafting operation.According to U.S.'s disease prevention and control centre's incomplete statistics, surpass 1,000,000 examples in U.S. every year because of the patient that the damaged needs of bone carry out the bone transplant operation, medical expense surpasses 5,000,000,000 dollars, has also surpassed 900,000,000 pounds in the expense of Britain.Traditional transplanting mode mainly contains autologous bone transplanting and allogenic bone transplantation, but autologous bone transplanting be faced with the bone source limited, increase defectives such as wound and infection rate, and allogenic bone transplantation exists that biological activity is poor, immunological rejection and risk of disease transmission.The damaged treatment of bone that develops into of tissue engineering technique in recent years provides new approaches.
Mesenchymal stem cells MSCs (MSCs) is easy to obtain and amplification because of having clear and definite Osteoblast Differentiation potential, and its unique cell proliferation schizotype makes exogenous gene be easy to import and express, and therefore becomes present research organizational project bone seeding cell the most widely.MSCs has skeletonization potential, and directionality osteoprogenitor cells wherein can be divided into osteoblast automatically on the one hand, and inductivity osteoprogenitor cells wherein also can be converted into osteoblast system under the effect of inducer on the other hand.Though a large amount of experiment confirms has the been arranged Osteoblast Differentiation of MSCs, how to make MSCs carry out the directional induction Osteoblast Differentiation by suitable genetic modification, thus better application is a problem demanding prompt solution in bone tissue engineer.Osteoblast idiosyncratic transcription factor-2 (Osf2), claim core binding factor α 1 (Cbfa1) again, the transcription factor that belongs to runt domain gene family, be proved osteoblast differentiation and bone formation have been had important function, but do not seen both at home and abroad Osf2 is applied to the report that tissue-engineered bone makes up.
In addition, in bone tissue engineer, good timbering material is beneficial to the location of cell and/or bioactie agent and sends, and influences the formation of neoblastic three dimensions configuration afterwards, and final adapt to well with receptor and combine, formation has the new organization of suitable 26S Proteasome Structure and Function.Ideal engineering material of bone tissue should satisfy following requirement: 1. excellent biological compatibility; 2. favorable biological degradability; 3. easily shape, and have certain intensity; 4. material surface is easy to cell adhesion and does not influence its proliferation and differentiation.Existing a large amount of experimental applications synthetic material carries out the damaged reparation of bone to be attempted, and obtains certain effect, but problems such as biocompatibility, catabolite metabolism, mechanical characteristic have limited its application.Therefore, seek a kind ofly to take all factors into consideration biocompatibility, biological degradability, osteoinductive and bone conductibility, and the material preparation method of mechanical property is very necessary.Natural biological derived material antigenicity a little less than, and favorable tissue affinity and adhesion are arranged, the pore structure that it is natural is for adhesion, propagation, differentiation and the skeletonization of seed cell provides natural three-D space structure.But present bio-derived material also has its drawback: deproteinization is handled pore structure and the mechanical strength that has kept material preferably, has reduced antigenicity, but has reduced the osteoinductive of material simultaneously; Keep inductivity preferably though decalcification is handled, greatly reduced mechanical strength.
Cell is a difficult point in the present bone tissue engineer research at the In vitro culture of biologic bracket material, main cause is that simple dimensional culture can cause cell distribution inhomogeneous, problems such as the nutrition exchange obstacle of deep cell, therefore using reasonable and practical external plantation and cultivating system is the key of constructing tissue through engineering approaches bone.
Summary of the invention
The present invention is directed in the research of present bone tissue engineer seed cell, timbering material and three aspects of In vitro culture and improve, purpose provides a kind of construction method with tissue-engineered bone of clear and definite bone-forming effect, is used for repairing bone defect clinically.
Technical solution of the present invention is as follows:
This method is a seed cell with the mesenchymal stem cells MSCs of osteoblast idiosyncratic transcription factor-2 genetic modification, with take off the compound structure osseous tissue of cell bone matrix natural biological derived material, after external dynamic cultivation, carry out the damaged reparation of big segment length's bone, comprise following three steps:
(1) obtaining of seed cell: described seed cell is a mesenchymal stem cells MSCs, utilizes osteoblast idiosyncratic transcription factor-2 (Osf2) adenovirus infection after the cell fusion degree is about 90%, infects collecting cell after 24 hours and is used for compound with timbering material;
(2) preparation of natural biological derived material: remove antigenicity and cell component by xenogenesis or osseous tissue of the same race, keep the natural hole shelf structure and the mechanical characteristic of bone matrix;
(3) the dynamic cultivation of the plantation of seed cell and tissue-engineered bone: after finishing the preparation of timbering material, on timbering material, inoculate seed cell.Then in reactor, dynamically cultivated 5-7 days, promptly obtain tissue-engineered bone.
Beneficial effect of the present invention:
The present invention is directed in the research of present bone tissue engineer seed cell, timbering material and three aspects of In vitro culture improves.By Osf2 genetic modification bone marrow MSCs, strengthened the skeletonization directed differentiation ability of seed cell, and in repair process, promoted bone formation; Take off the application of cell bone matrix bio-derived material, only slough cell component, and the inorganic and organic principle of reservation tissue, the utmost point is beneficial to the formation of adhering to of cell and new bone, makes timbering material comparatively desirable aspect biocompatibility, biological degradability, osteoinductive, bone conductibility and mechanical property; Dynamically cultivate and promoted seed cell at the sticking of timbering material, growing multiplication, and further promote the Osteoblast Differentiation ability of the MSCs of Osf2 genetic modification, to have solved cell be a difficult problem in the present bone tissue engineer research at the In vitro culture of biologic bracket material.The present invention is applicable to multiple common reparations such as orthopaedics, shaping, decorative sursery, has broad clinical application prospect.
Description of drawings
Fig. 1 is the subcutaneous plantation in tissue-engineered bone nude mice back among the embodiment 1.The 4 visible a large amount of new osteogenesis in week back.Fluorescent tracing observation and histological examination are found, a large amount of seed cell performance bone-forming effects (200 *) through genetic modification in the tissue-engineered bone.
Fig. 2. be the damaged reparation of rabbit radius 1.2cm among the embodiment 2.The X ray examination: postoperative 12 all A pack support materials are degraded fully and are replaced by freshman bone tissue, and newly bone is moulding finishes, and medullary cavity is unobstructed.The A group obviously is better than other group at aspects such as new bone formation, bone remodeling and pulp cavity are logical again.
The specific embodiment
Describe building process of the present invention in detail below in conjunction with embodiment:
Embodiment 1:
1. the preparation of seed cell:
Extract the about 2m1 of mouse bone marrow cells suspension,, collect the mononuclear cell layer at interface, be adjusted to 2 * 10 by density gradient centrifugation
5/ cm
2Density inoculation.Change liquid after 24 hours first, abandon or adopt not attached cell, after this half amount is changed liquid 2 times weekly.Cell monolayer merges substantially behind the 14d.0.25% trypsinization went down to posterity by 1: 2.Use 1-6 for cell, utilize the adenovirus infection of carrier Osf2 gene after the cell fusion degree is about 90%, infect collecting cell after 1 day and be used for compound with timbering material.
1.1 obtaining of mesenchymal stem cells MSCs: extract the about 2ml of bone marrow suspension from the mice femur, slowly add in the centrifuge tube that adds Percoll separating medium 5mL (proportion 1.073g/mL) in advance, centrifugal with 2000r/min * 20min, collect the mononuclear cell layer at interface, PBS washes 2 times, removes superabundant fats and tissue fluid, add the DMEM culture fluid, piping and druming is diluted to cell suspension, is adjusted to 2 * 10
5/ cm
2Density be inoculated in the 25ml culture bottle.Add the capacity culture fluid, culture medium is DMEM+10%FCS, 5%CO
2, hatch for 37 ℃, change liquid behind the 24h, abandon or adopt not attached cell, after this half amount is changed liquid 2 times weekly, observes day by day.Cell monolayer merges substantially behind the 14d.0.25% trypsinization went down to posterity by 1: 2.Collect 1-6 and be used for following structure for cell.
1.2 carrier Osf2 adenovirus vector construct is as follows: the design primer, the upstream:
5 '-
GAAGATCTTCATGGCATCAAACAGCCTCTT-3 ' (adding the BgllI restriction enzyme site); The downstream
5 '-
GCCTCGAGCGTCAATATGGTCGCCAAACAGATTCA-3 ' (adding the XhoI restriction enzyme site), the total RNA of extracting osteoblast, with RT-PCR method human cloning Osf2 gene cDNA, directed cloning is in BglII (947bp) and XbaI (972bp) site of shuttle plasmid pShuttle-CMV (the numbering #240007 of U.S. Stratagene company), then utilize antibacterial interior reorganization principle and skeleton plasmid pAdeasy-1 (the numbering #240005 of U.S. Stratagene company) reorganization to obtain the pAdEasy-1/Osf2 plasmid, liposome transfection 293 cells, recombinant adenovirus is in the breeding of increasing in a large number of 293 cells, cesium chloride density gradient ultracentrifugation purification obtains the Osf2 recombinant adenovirus.
With above-mentioned constructed recombinant adenovirus genomic DNA is template, can amplify the Osf2 gene segment, and fail to amplify the specific band of adenovirus E 1 protein, the adenovirus that shows acquisition carries the Osf2 gene, and do not have the adenovirus that possesses replication capacity and pollute, can be applied to the genetic modification of seed cell.
1.3 the concrete operations of Osf2 adenovirus infection mesenchymal stem cells MSCs are as follows: with MSCs with 2 * 10
4/ cm
2Density go down to posterity in 6 orifice plates, cell fusion is about 90% o'clock behind about 3d, wash 2 times with PBS, the Osf2 recombinant adenovirus that adds infection multiplicity (MOI)=50, infect that PBS washes 2 times after 1 hour, add conditioned medium (DMEM+10% hyclone+dexamethasone 10nmol/l+ vitamin C 50mg/l+ sodium 10mmol/L).
2. the preparation method of natural biological derived material in the following ways: xenogenesis or spongy bone piece of the same race are inserted in 1: 2 methanol chloroformic solution in defat 12h → 0.05M Tris-HCl (PH7.4) buffer, add protease inhibitor (4U/mlDispase in the buffer, l μ M Acetyl-Pepstain, 0.5 μ M AEBSF, 2 μ g/ml Aprotinins, 100 μ M Leupeptin), 4 ℃ of isothermal vibration 3d → bone pieces are inserted in Tris-HCl (PH7.4) buffer that contains 3%TritonX-100, in add protease inhibitor, after 4 ℃ of isothermal vibration 3d → deionized waters wash 12h continuously, digest 12h → the 3rd step of repetition under adding DNAase and the RNAase mixed liquor room temperature.Last bone piece is after distilled water fully washes, and vacuum system is done, through the 10kGy cobalt
60-20 ℃ of preservations are standby behind the-radiation gamma sterilization.
As seen to take off cell bone matrix arrangement of collagen fibers neat with cell extracellular matrix material row morphologic detection: the HE dyeing of taking off of method for preparing, and trabecular bone structure is complete, does not have obviously fracture, and is hollow in the bone lacuna, do not see cell and other cell component.There is no cell in the lacuna around the haversian system central canal reach, whole substrate is Yihong color.It is identical that edge part and central part take off cell effect.Scanning electron microscope shows: it is more complete to take off cell bone matrix structure, the bone lacuna inanition, and lacuna inner wall smooth rule is not seen any cell residue composition.Taking off the mesh of cell bone matrix measures: take off the cell bone matrix and present irregular rack-gap structure, through measuring 500 holes in 50 visuals field, the pore diameter that takes off the cell bone matrix by statistics is 253 ± 106 μ m, porosity about 75.8%.
The mechanical index of this timbering material: several indexs such as the compression failure load of bone, maximum compressive strength, elastic modelling quantity slightly reduce and reduce, but not statistically significant (P＞0.05), it is the not significantly change of mechanical property of compression vertically.Heteroantigen α-gal does not have expression.
3. the dynamic cultivation of the plantation of seed cell and tissue-engineered bone: actual example is: 1. will take off the cell bone matrix (2h that prewets in the DMEM culture medium of 1.0cm * 1.0cm * 0.5cm).2. then with 2.0 * 10
6Individual MSCs through the Osf2 adenovirus infection makes 400 μ l cell suspension, utilizes the 1ml syringe to add host material, and cell is poured in the hole.24 orifice plates leave standstill cultivation, and 37 ℃, 5%CO
2Incubator was cultivated 4 hours.3. the last DMEM culture medium that contains 10%FCS that adds covers host material, overnight incubation (12-16 hour).Change liquid weekly 2 times.4.: at the cell perfusion system of design preparation voluntarily, perhaps rotary cell culture system (RCCS) etc. carries out the dynamic cultivation of tissue-engineered bone.Cell-material composite is placed flow cavity, adjust the position, make the culture medium timbering material of can fully flowing through, cultivated 5-7 days, change liquid weekly 2 times.
Cultivation 4 hours is left standstill in the plantation of carrying out seed cell through above-mentioned steps, after but the culture medium culturing that adds cladding material spent the night, inverted microscope was observed down, can find that material hole and periphery have cell adhesion, along with the time lengthening of cell culture, the cell that is attached on the material increases gradually.Electronic Speculum shows: cultivate after 5 days in reactor, cell adheres in material well, and cellular morphology is fibrous fusiformis or flat, stretches out hole or the surface of cell process " riveting volt " at material.Have projection to be connected between the flanking cell, cell surface has the calcium particle deposition.The alkaline phosphatase activities of cell and Bone Gla protein product all are higher than the static culture group and organize without Osf2 genetic modification MSCs, show at fluid and promote on the basis of cell differentiation, genetic modification by Osf2, further enhanced MSC s is to osteoblastic differentiation, thereby better brings into play the repair function of tissue-engineered bone.
Carry out external dynamic cultivation after 5 days according to step 3, the subcutaneous plantation in nude mice back.The 4 visible a large amount of new osteogenesis in week back.See Fig. 1, fluorescent tracing observation and histological examination are found, a large amount of seed cell performance bone-forming effects (200 *) through genetic modification in the tissue-engineered bone.
Embodiment 2:
Step 1 method of pressing among the embodiment 1 obtains the rabbit bone marrow mescenchymal stem cell, behind the Osf2 genetic modification, is planted in set by step 2 prepared timbering materials, carries out external dynamic cultivation 5 days according to step 3 then.Select 40 of 3 monthly age Japan large rabbits, it is damaged to set up one-sided radius 1.2cm, is divided into 4 groups according to different repair modes, 10 every group: A organizes .Osf2 genetic modification MSCs and takes off the compound back reparation of cell extracellular matrix material; B group. not genetic modification MSCs with take off the compound back of cell extracellular matrix material and repair; The C group. simple timbering material reparation; The D group. blank does not add any bone grafting and handles.The result is referring to Fig. 2, X ray examination and histological observation show that the A group obviously is better than other group at aspects such as new bone formation, bone remodeling and pulp cavity are logical again, postoperative 12 all A pack support materials are degraded fully and are replaced by freshman bone tissue, and newly bone is moulding finishes, and medullary cavity is unobstructed; Repair back reparation side radius and compare destruction compressive load difference not remarkable (P＞0.05) with strong side.
If said method is for the people, seed cell is a mesenchymal stem cells MSCs of taking from anterior superior iliac spine, and all the other implementation steps are identical.
Claims (9)
1. the construction method of the tissue-engineered bone of osteoblast idiosyncratic transcription factor-2 genetic modification is characterized in that: adopt following method preparation:
(1) obtaining of seed cell: described seed cell is a mesenchymal stem cells MSCs, utilizes osteoblast idiosyncratic transcription factor-2 (Osf2) adenovirus infection after the cell fusion degree is about 90%, infects collecting cell after 24 hours and is used for compound with timbering material;
(2) preparation of natural biological derived material: remove antigenicity and cell component by xenogenesis or osseous tissue of the same race, keep the natural hole shelf structure and the mechanical characteristic of bone matrix;
(3) the dynamic cultivation of the plantation of seed cell and tissue-engineered bone: after finishing the preparation of timbering material, on timbering material, inoculate seed cell, in reactor, dynamically cultivated 5-7 days then.
2. the construction method of the tissue-engineered bone of osteoblast idiosyncratic transcription factor-2 genetic modification according to claim 1, it is characterized in that, in its preparation method (1) mesenchymal stem cells MSCs obtain as follows: extract the about 2ml of bone marrow suspension, slowly add in the centrifuge tube that adds Percoll separating medium 5mL in advance, centrifugal with 2000r/min * 20min, collect the mononuclear cell layer at interface, with 2 * 10
5/ cm
2Density be inoculated in the 25ml culture bottle; Change liquid behind the 24h, abandon or adopt not attached cell, after this half amount is changed liquid 2 times weekly, observes day by day; Cell monolayer merges substantially behind the 14d; 0.25% trypsinization went down to posterity by 1: 2.
3. the construction method of the tissue-engineered bone of osteoblast idiosyncratic transcription factor-2 genetic modification according to claim 1 is characterized in that, carrier Osf2 gene adenovirus vector construct is as follows in its preparation method (1): design primer upstream: 5 '-
GAAGATCTTCATGGCATCAAACAGCCTCTT-3 '; Downstream 5 '-
GCCTCGAGCGTCAATATGGTCGCCAAACAGATTCA-3 ', the total RNA of extracting osteoblast, with RT-PCR method human cloning Osf2 gene cDNA, directed cloning is in BglII and the XbaI site of shuttle plasmid pShuttle-CMV, afterwards utilize antibacterial interior reorganization principle and skeleton plasmid pAdeasy-1 reorganization to obtain the pAdEasy-1/Cbfal plasmid, liposome transfection 293 cells, recombinant adenovirus is at the breeding of increasing in a large number of 293 cells, purification.
4. the construction method of the tissue-engineered bone of osteoblast idiosyncratic transcription factor-2 genetic modification according to claim 1, it is characterized in that the concrete operations of Osf2 adenovirus infection mesenchymal stem cells MSCs are as follows in its preparation method (1): with MSCs with 2 * 10
4/ cm
2Density go down to posterity in 6 orifice plates, cell fusion is about 90% o'clock behind about 3d, adds the Osf2 recombinant adenovirus of infection multiplicity (MOI)=50, infects that PBS washes 2 times after 1 hour, the adding conditioned medium.
5. the construction method of the tissue-engineered bone of osteoblast idiosyncratic transcription factor-2 genetic modification according to claim 4, it is characterized in that the conditioned medium that adds in the preparation method (1) is: DMEM+10% hyclone+dexamethasone 10nmol/L+ vitamin C 50mg/L+ sodium 10mmol/L.
6. the construction method of the tissue-engineered bone of osteoblast idiosyncratic transcription factor-2 genetic modification according to claim 1, it is characterized in that, the preparation method of natural biological derived material is as follows in its preparation method (2): xenogenesis or spongy bone piece of the same race are inserted in 1: 2 methanol chloroformic solution in defat 12h → 0.05M Tris-HCl buffer, PH7.4, add protease inhibitor in the buffer, the 4 ℃ of isothermal vibration 3d → bone piece is inserted in the Tris-HCl buffer that contains 3%TritonX-100, PH7.4, in add protease inhibitor, after 4 ℃ of isothermal vibration 3d → deionized waters wash 12h continuously, digest 12h → the 3rd step of repetition under adding DNAase and the RNAase mixed liquor room temperature; Last bone piece is after distilled water fully washes, and vacuum system is done, through the 10kGy cobalt
60-20 ℃ of preservations are standby behind the-radiation gamma sterilization.
7. the construction method of the tissue-engineered bone of osteoblast idiosyncratic transcription factor-2 genetic modification according to claim 6, it is characterized in that, the protease inhibitor that adds in the preparation method (2) is: 4U/mlDispase, 1 μ MAcetyl-Pepstain, 0.5 μ M AEBSF, 2 μ g/ml Aprotinins and 100 μ M Leupeptin.
8. the construction method of the tissue-engineered bone of osteoblast idiosyncratic transcription factor-2 genetic modification according to claim 1 is characterized in that: the plantation of seed cell operation is as follows in its preparation method (3): at first will take off the cell bone matrix and prewet in the DMEM culture medium 2 hours; Then with 2.0 * 10
6Individual MSCs through the Osf2 adenovirus infection makes 400 μ l cell suspension, utilizes the 1ml syringe to add host material, and cell is poured in the hole; 24 orifice plates leave standstill cultivation, and 37 ℃, the 5%CO2 incubator is cultivated 4h; Add the DMEM culture medium that contains 10%FCS at last and cover host material, cultivate 12-16h, change liquid weekly 2 times.
9. the construction method of the tissue-engineered bone of osteoblast idiosyncratic transcription factor-2 genetic modification according to claim 1, it is characterized in that, the dynamic cultivation of tissue-engineered bone in its preparation method (3): tissue-engineered bone is dynamically cultivated in reactor, utilize cell perfusion system or rotary cell culture system RCCS, cell-material composite is placed flow cavity, adjust the position, make the culture medium timbering material of can fully flowing through, cultivated 5-7 days, and changed liquid weekly 2 times.
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| JPH05268982A (en) * | 1992-03-27 | 1993-10-19 | Hoechst Japan Ltd | Novel protein having osteogenic action and its production |
| CN1146368C (en) * | 2000-12-15 | 2004-04-21 | 四川大学华西医院 | Biologically derived tissue engineering bone and preparation method thereof |
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