CN100418582C - Microcapsule for promoting bone regeneration and its use - Google Patents
Microcapsule for promoting bone regeneration and its use Download PDFInfo
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- CN100418582C CN100418582C CNB2005101107505A CN200510110750A CN100418582C CN 100418582 C CN100418582 C CN 100418582C CN B2005101107505 A CNB2005101107505 A CN B2005101107505A CN 200510110750 A CN200510110750 A CN 200510110750A CN 100418582 C CN100418582 C CN 100418582C
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Abstract
The present invention relates to a microcapsule for promoting bone regeneration, which is prepared by the following methods: (1), the carrying of recombinant adenoviruses of bone morphogenetic generation protein genes, (2), the cultivation of bone marrow mesenchymal stem cells, (3), the carrying of the bone marrow mesenchymal stem cells of adenovirus vector transfection of bone generation genes, and (4), the microencapsulation of the bone marrow mesenchymal stem cells of bone morphogenetic generation protein gene transfection. The present invention has the advantages that non-autologous stem cells are allowed to be used as gene transfection target cells; in this way, the time of amplifying and culturing autologous stem cells is reduced; the stem cells are used for emergency patients and can be screened outside bodies for ensuring cell quality and enlarging the application ranges of the cells and gene therapy.
Description
Technical field
The present invention is about a kind of microcapsule, especially in regard to a kind of microcapsule that promotes osteanagenesis.
Background technology
The gene therapy of osteanagenesis at present utilizes autologous stem cells as the gene transfection stem cell, as the bone morphogenesis protein-2 gene that utilizes adenovirus vector to carry, and the transfection autologous bone marrow mesenchymal stem cells is replanted defect to the marrow, promotes osteanagenesis.All achieve success on one's body at big toy at present.But present osteanagenesis gene therapy also has following shortcoming: (1) all utilizes the target cell of autogenous cell as gene transfection, because separation, cultivation, the amplification of cell need the long period, can't be used for the emergency case; Even for the patient who chooses date for operation, need patient at first to come institute to get bone marrow, treat that cell culture arrives requirement and (in our experience, obtains 10
7The cell 25 days left and right sides time of needs) come hospital again after, be difficult to be accepted by patient.Moreover individual stem cell population and quality exist than big-difference, is difficult to guarantee that each patient's of being cultivated cell all has vigor preferably.Quality and quantity as stem cell in old people's bone marrow is all poor than youngster, and the patient who suffers from systemic disease, metabolic disease and chemotherapy faces the problem of stem cell population deficiency, downgrade too.(2) as adopting the carrier of adenovirus as gene, adenovirus itself can be induced rejection, has limited genes of interest expression time in vivo.(3) as utilizing allosome or heterogenous cell as genetically modified target cell, need to use the general immunosuppressant, the immunologic function of human body there is certain infringement.
Summary of the invention
The object of the present invention is to provide:
(1) a kind of microcapsule that promotes osteanagenesis;
(2) microcapsule is used in preparation treatment osteopathia medicine.
The objective of the invention is to realize by following technical method:
(1) carries the recombinant adenovirus of bone morphogenetic protein gene, BMP-2cDNA fragment (being provided by ATCC) is inserted among the carrier PAC-CMV, constitute recombiant plasmid PAC-CMVBMP2, again with PAC-CMVBMP2 through behind the Xho I enzyme action with the big fragment of 5 type adenoviral genes behind Cla I enzyme action homologous recombination in 293 cells, form the recombinant adenovirus carry bone morphogenetic protein gene-2 gene.
(2) cultivation of rat bone marrow mesenchymal stem cells and Adenovirus Transfection are got body weight 120g left and right sides SD rat, the ketamine intraperitoneal anesthesia.Under the aseptic condition of strictness, isolate the bilateral femur, with its far and near end of rongeur amputation.Flush out myeloid tissue with 10ml alpha-MEM complete medium, be inoculated in the 100mm culture dish, every ware 10ml places 37 ℃, 5%CO
2Incubator is hatched.Later half amount was changed liquid in 3 days, changed liquid after 1 week fully, changed liquid later on weekly 2 times.On 14 days, attached cell almost 90% converged, and the exhaustion culture medium with PBS flushing 2 times, adds 1ml 0.05 trypsin and contains 0.02%EDTA), place 37 ℃, 5%CO
2Incubator was hatched 5 minutes, added 9ml low sugar a-MEM complete medium with the tryptic activity that neutralizes.Blow and beat cell gently with suction pipe and make and become single cell suspension, by the cultivation of going down to posterity in 1: 3, the cell of this moment claimed first passage cell.Carrying out the 2nd passage cell with method cultivates.After the 2nd passage cell nearly 90% converges, blot culture medium, every ware adds 4ml virus working solution and spends the night (MOI=100), or does not add processing, and hatching is spent the night.The viral working solution that exhausts, every ware add 10ml alpha-MEM complete medium (FCS is through hot deactivation) to be continued to cultivate.
(3) microencapsulation of the mesenchymal stem cells MSCs of BMP-2 gene transfection
The mesenchymal stem cells MSCs of BMP-2 gene transfection is suspended in 1.5% sodium alginate soln of filtration sterilization cell concentration 3 * 10 again
6/ ml.The cell suspending liquid of sodium alginate oozes by No. 7 blunt nosed pins of syringe pump, by large power high voltage pulse microcapsule preparing instrument (referring to utility model patent: microcapsule shaped device (patent No.: 00218100.2)), shear the drop that syringe pump oozes, and control its particle diameter.The sodium alginate Cell sap drops in gelation in the 100mM calcium chloride solution, forms calcium alginate cell glue pearl after about 5 minutes; After normal saline cleaning 2 times, calcium alginate cell glue pearl is suspended in the 0.05%-0.1% polylysine solution of filtration sterilization 5 minutes, polylysine and calcium alginate generation polyelectrolyte complex reaction film forming form embedding mesenchymal stem cells MSCs polylysine alginate microcapsule.The reuse normal saline cleans 2 times, immerses 0.15% sodium alginate soln afterwards about 5 minutes, forms another layer sodium alginate coat, cleans 2 times with normal saline, and with 55mM sodium citrate solution 20ml reaction 6 minutes, the core of microcapsule was liquefied then.After normal saline cleans, add culture medium, 37 ℃ of incubators are cultivated.
The present invention uses the ultimate principle of microencapsulation technology: utilize the chemical method embedding to transplant and use cell, form complete cyst membrane, i.e. microcapsule in the extracellular.Microcapsule has permselectivity, and hole on the film allows small-molecule substance such as nutrient substance to free in and out, but macromolecular substances such as antibody and immunocyte are blocked in outside the capsule.Microcapsule has been for its inside provides the microenvironment of an immunoprotection, make the cell heteroplastic transplantation in the microcapsule after, can avoid the attack of host's rejection, and at allosome survival justacrine therapeutic recombiant protein (general molecular weight is less than 100kD).Therefore, use the microencapsulation technology in cell transplantation, not only do not need to use immunosuppressant, also enlarged the source of transplantation donor, also bone is damaged have been saved the time in order to treat simultaneously.
The present invention: a kind of microcapsule that promotes osteanagenesis is made by following method:
(1) adenovirus vector carries the bone morphogenetic protein gene;
(2) cultivation of mesenchymal stem cells MSCs;
(3) carry the adenovirus vector transfection mesenchymal stem cells MSCs of skeletonization gene;
(4) microencapsulation of the mesenchymal stem cells MSCs of bone morphogenetic protein gene transfection.
The bone morphogenetic protein gene is a bone morphogenetic protein gene 2 in step (1) and the step (4).
Step (2) bone marrow is selected from rat marrow.
Flush out myeloid tissue with the alpha-MEM complete medium in the step (2).
Mesenchymal stem cells MSCs is suspended in the sodium alginate soln treated formation calcium alginate cell glue pearl in the step (4).In calcium alginate cell glue pearl suspension polylysine solution, treated formation embedding bone marrow stroma stem cell polylysine alginate microcapsule.
The disclosed a kind of microcapsule that promotes osteanagenesis of the present invention, its advantage shows: allow to use non-from the target cell of body (allosome or xenogenesis) stem cell as gene transfection, reduce like this and take from the time that the soma cell needs amplification cultivation, be used for the emergency case, and can be earlier at in-vitro screening, can guarantee the quality of cell, enlarge the range of application of cell and gene therapy.
Description of drawings
Fig. 1 shows: the microencapsulation of the mesenchymal stem cells MSCs of BMP-2 gene transfection.
Fig. 2 A shows: microencapsulation the 4th day, the expression of transgenic cell in the microcapsule is determined in external X-gal dyeing.
Fig. 2 B shows: microencapsulation the 28th day, the expression of transgenic cell in the microcapsule is determined in external X-gal dyeing.
Fig. 3 shows: the MSC microcapsule external secretion situation of transfection BMP-2.5 groups every, average.Every group of microcapsule inner cell total amount about 2.5 * 10
6/ group is incubated at 15ml alpha-MEM (containing 15% calf serum).
The specific embodiment
Below in conjunction with embodiment the present invention is further described.
Embodiment 1
The cultivation of rat bone marrow mesenchymal stem cells and Adenovirus Transfection
Get body weight 120g left and right sides SD rat, the ketamine intraperitoneal anesthesia.Under the aseptic condition of strictness, isolate the bilateral femur, with its far and near end of rongeur amputation.Flush out myeloid tissue with 10ml alpha-MEM complete medium, be inoculated in the 100mm culture dish, every ware 10ml places 37 ℃, 5%CO
2Incubator is hatched.Later half amount was changed liquid in 3 days, changed liquid after 1 week fully, changed liquid later on weekly 2 times.On 14 days, attached cell almost 90% converged, and the exhaustion culture medium with PBS flushing 2 times, adds 1ml 0.05 trypsin and contains 0.02%EDTA), place 37 ℃, 5%CO
2Incubator was hatched 5 minutes, added 9ml low sugar a-MEM complete medium with the tryptic activity that neutralizes.Blow and beat cell gently with suction pipe and make and become single cell suspension, by the cultivation of going down to posterity in 1: 3, the cell of this moment claimed first passage cell.Carrying out the 2nd passage cell with method cultivates.After the 2nd passage cell nearly 90% converges, blot culture medium, every ware adds 4ml virus working solution and spends the night (MOI=100), or does not add processing, and hatching is spent the night.The viral working solution that exhausts, every ware add 10ml alpha-MEM complete medium (FCS is through hot deactivation) to be continued to cultivate.
The microencapsulation (see figure 1) of the mesenchymal stem cells MSCs of BMP-2 gene transfection
The mesenchymal stem cells MSCs of BMP-2 gene transfection is suspended in 1.5% sodium alginate soln of filtration sterilization cell concentration 3 * 10 again
6/ ml.The cell suspending liquid of sodium alginate oozes by No. 7 blunt nosed pins of syringe pump, by large power high voltage pulse microcapsule preparing instrument (referring to utility model patent: microcapsule shaped device (patent No.: 00218100.2)), shear the drop that syringe pump oozes, and control its particle diameter.The sodium alginate Cell sap drops in gelation in the 100mM calcium chloride solution, forms calcium alginate cell glue pearl after about 5 minutes; After normal saline cleaning 2 times, calcium alginate cell glue pearl is suspended in the 0.05%-0.1% polylysine solution of filtration sterilization 5 minutes, polylysine and calcium alginate generation polyelectrolyte complex reaction film forming form embedding mesenchymal stem cells MSCs polylysine alginate microcapsule.The reuse normal saline cleans 2 times, immerses 0.15% sodium alginate soln afterwards about 5 minutes, forms another layer sodium alginate coat, cleans 2 times with normal saline, and with 55mM sodium citrate solution 20ml reaction 6 minutes, the core of microcapsule was liquefied then.After normal saline cleans, add culture medium, 37 ℃ of incubators are cultivated.
Embodiment 3
The expression (seeing Fig. 2 A, Fig. 2 B) of transgenic cell in the microcapsule is determined in external X-gal dyeing
For the effectiveness in external assessment gene therapy, the most primary is exactly whether the gene of seeing institute's transfection expresses.For this reason, we first transfection reporter gene β-gal goes into MSC, microencapsulation again, and whether observe the microcapsule inner cell can continuous release β-gal.The MSC of transfection β-gal behind microencapsulation 7 days 14,21,28 days, uses the dyeing of X-gal group respectively.At first microcapsule is moved in the 15ml centrifuge tube, fix 10 minutes with 0.5% glutaraldehyde, PBS gives a baby a bath on the third day after its birth time, adds 37 ℃ of 5%CO of X-gal dye liquor again
2Incubator spends the night.The X-gal dye liquor contains 1mg/ml X-gal, 35mM K
3Fe (CN)
6, 35mMK
3Fe (CN)
6.3H
2O, 2mM MgCl
2. put into the observation of 10cm culture dish microscopically next day and take pictures.The result shows, the commentaries on classics β in the microcapsule-gal cell can be the same with the commentaries on classics β-gal cell of microcapsule not, excreting beta-gal.And at least can continuous release 28 days.
ELISA measures the content (see figure 3) of secreting the gene recombinaton product B MP2 to capsule
When changing liquid, left and took supernatant behind the mesenchymal stem cells MSCs microencapsulation of transfection BMP-2 gene every 2 days,, measure the content of BMP2 with the ELISA method.Take out the special-purpose orifice plate of ELISA, every hole adds 100ulAssay Diluent RD-19 earlier, adds standard substance or sample again, seals with sealing, and shaking table is 2 hours under the room temperature.With 400ul Wash Buffer hole flushing, wash firmly clappers of back, repeat to wash plate 4 times, every again hole crust 200ulBMP-2 Conjugate room temperature shaking table 2 hours, repeat hole flushing again 4 times, add 200ulSubstrate Solution again, the lucifuge room temperature is after following 30 minutes, add 50ul StopSolution, survey OD450nm. behind the mixing under the microplate reader and do blank with the MSC culture supernatant of untransfected BMP-2 gene simultaneously.The result shows, surveys the content of BMP-2 in nearly one month time continuously.Compare obvious difference with the blank group.Showing equally can continuous release BMP-2 in the microcapsule.
Embodiment 5
The excretory BMP2 determination of activity of transgenic cell (seeing Table 1) in the microcapsule
In order to verify the activity of the endocrine BMP-2 of microcapsule, we are microcapsule (every hole microcapsule inner cell 1 * 10
6) cultivate altogether with mesenchymal stem cells MSCs, observe the conversion situation of the outer MSC of capsule.MSC microcapsule+MSC that MSC microcapsule+MSC of grouping (1) untransfected BMP-2 cultivates (2) commentaries on classics rhBMP-2 altogether cultivates altogether, row alkali phosphatase (ALP after 8 days, be osteoblastic marker protein) quantitative assay, add VitC (50 μ g/ml) in the culture medium, changed 1/3 culture medium in per 2 days, culture medium exhausted after 8 days, with Tris buffer salt solution flushing three times, the Tris that every ware adds 0.5ml 50mmol/L scrapes collection with cell, is positioned over-20 ℃ of frozen surveys fully.During detection, thaw under the room temperature, cell is measured the concentration of ALP in the lysate through ultrasonic degradation with ALP detection by quantitative test kit (Sigma).The sample number of each group is 5.The result shows that the ALP activity of mesenchymal stem cells MSCs is apparently higher than non-transfected cells microcapsule group, p＜0.05 in the Adv-hBMP-2 transfectional cell microcapsule group.
Table 1 respectively organize ALP activity (sigma unit/ml lysate) (mean ± standard deviation, IU)
There was a significant difference for p＜0.05 a liang group
Claims (3)
1. microcapsule that promotes osteanagenesis is made by following method:
(1) carries the adenovirus vector of bone morphogenetic protein gene;
(2) cultivation of mesenchymal stem cells MSCs;
(3) carry the adenovirus vector transfection mesenchymal stem cells MSCs of bone morphogenetic protein gene;
(4) microencapsulation of the mesenchymal stem cells MSCs of bone morphogenetic protein gene transfection, described mesenchymal stem cells MSCs concentration is 3 * 10
6/ ml, this cell suspension is in 1.5% sodium alginate soln, the bone morphogenetic protein gene is the human bone morphogenesis protein-2 gene in described step (1) and the step (4), flush out myeloid tissue with the alpha-MEM complete medium in the step (2), mesenchymal stem cells MSCs is suspended in the sodium alginate soln in the step (4), treated formation calcium alginate cell glue pearl, in calcium alginate cell glue pearl suspension polylysine solution, treated formation embedding mesenchymal stem cells MSCs polylysine alginate microcapsule.
2. microcapsule according to claim 1 is characterized in that: step (2) bone marrow is selected from rat marrow.
3. the described microcapsule of claim 1 is used in preparation treatment osteopathia medicine.
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| CN101564555B (en) * | 2009-05-27 | 2013-01-23 | 深圳市第二人民医院 | Tissue engineering bone implant and method for constructing the same |
| CA3025308A1 (en) * | 2016-05-24 | 2017-11-30 | The Regents Of The University Of California | Growth-factor nanocapsules with tunable release capability for bone regeneration |
| CN111330072B (en) * | 2020-03-03 | 2021-11-23 | 南京鼓楼医院 | Preparation method and application of bionic porous MSCs microspheres |
| CN116769725A (en) * | 2023-08-18 | 2023-09-19 | 苏州大学附属第二医院 | RANK-overexpressing MSCs extracellular vesicle vector, and preparation method and application thereof |
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| CN1335148A (en) * | 2000-07-26 | 2002-02-13 | 郭德荣 | Osteanaphysis capsule for treating femoral head necrosis |
| CN1353185A (en) * | 2000-11-02 | 2002-06-12 | 上海兆安医学科技有限公司 | Recombinant adenovirus of bone morphogenetic protein and its method for exciting bone generation |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1335148A (en) * | 2000-07-26 | 2002-02-13 | 郭德荣 | Osteanaphysis capsule for treating femoral head necrosis |
| CN1353185A (en) * | 2000-11-02 | 2002-06-12 | 上海兆安医学科技有限公司 | Recombinant adenovirus of bone morphogenetic protein and its method for exciting bone generation |
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