CN104524636A - Method for obtaining tissue engineering cartilage through directionally inducing bone-marrow mesenchymal stem cells (BMSCs) - Google Patents
Method for obtaining tissue engineering cartilage through directionally inducing bone-marrow mesenchymal stem cells (BMSCs) Download PDFInfo
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Abstract
The invention relates to a method for obtaining tissue engineering cartilage through directionally inducing bone-marrow mesenchymal stem cells (BMSCs). At present, a large amount of exogenous growth factors and culture in vitro are needed before culturing the BMSCs and the cartilage cells in vivo to promote the differentiation of the BMSCs towards the cartilage cells. The method comprises the following steps of: mixing the sub-cultured P3-generation BMSCs, the cartilage cells and a large extracellular matrix aggregate (cell brick), resuspending the mixture into a platelet rich plasma to prepare a cell compound of the sub-cultured P3-generation BMSCs, the cell brick and the platelet rich plasma, injecting the compound into the skin on the back of a nude mice when the compound condenses gradually, and culturing to obtain the new tissue engineering cartilage. The completely biological and injectable tissue engineering cartilage is obtained on the basis of coculturing the BMSCs and the cartilage cells. The differentiation of the BMSCs towards the cartilage cells can be kept stably, the hypertrophy of the tissue engineering cartilage in vivo can be inhibited effectively, and the defects in the background art can be overcome.
Description
Technical field
The present invention relates to a kind of method building tissue engineering bone/cartilage, be specifically related to a kind of method being obtained tissue engineering bone/cartilage by directional induction mesenchymal stem cells MSCs.
Background technology
How building injectable, simultaneously to have continuous stability cartilage phenotype tissue engineering bone/cartilage, is a major challenge that clinical cartilage defect repair faces.Mesenchymal stem cells MSCs (BMSCs), has cartilage to differentiation potential.As BMSCs and chondrocyte Dual culture, can promote that the cartilage of BMSCs is to differentiation.Current promotion BMSCs cartilage before carrying out culturein vivo, needs a large amount of exogenous growth factor and In vitro culture operation to the method for breaking up.But how to suppress BMSCs hypertrophyization and then ossified generation in vivo, maintain continual and steady cartilage and remain bottleneck problem in clinical practice to differentiation.
Summary of the invention
The object of this invention is to provide and a kind ofly obtain the method for tissue engineering bone/cartilage by directional induction mesenchymal stem cells MSCs, obtains completely biological, injectable, can stable maintenance BMSCs cartilage to the tissue engineering bone/cartilage broken up.
The technical solution adopted in the present invention is:
Obtained the method for tissue engineering bone/cartilage by directional induction mesenchymal stem cells MSCs, it is characterized in that:
Realized by following steps:
Step one: the preparation of chondrocyte and the large aggregation of extracellular matrix:
(1) chondrocyte isolation, becomes Membrance cuiture:
That gets 1 monthly age New Zealand white rabbit enters Ear cartilage, and shredding rear mass fraction is that the II Collagenase Type solution of 0.2% is 37
zerodigest 12 hours at C temperature, afterwards, get single cell suspension in 15ml centrifuge tube with the centrifugal 5min of the rotating speed of 1000rpm;
Supernatant discarded, PBS rinses 1 time, with the centrifugal 5min of 1000rpm rotating speed, utilizes film forming induced liquid re-suspended cell, with 6.5 × 10
5individual cell/cm
2concentration plant and cultivate in 6 orifice plates, within every 3 days afterwards, change a not good liquor, culture fluid and aforementioned film forming induced liquid;
(2) chondrocyte diaphragm cell bricking process:
After 10 days, chondrocyte film forming, scrapes chondrocyte diaphragm, and it is pave in the culture dish of the sodium alginate solution of 1.2% that immersion fills mass fraction, the calcium chloride solution of instillation 102mM is until sodium alginate solution is condensed into gelatin complex completely, and thickness is 3-5mm;
Cut gelatin complex with the first horizontal rear rip cutting of multi-disc cutter, and be dipped in alginate dissolving buffer, release obtains cell brick; PBS rinses twice, for subsequent use;
Step 2: platelet rich plasma extracts:
Get 2 monthly age New Zealand white rabbit auricular vein blood, add by 10% of cumulative volume the sodium citrate solution anticoagulant that mass fraction is 3.8%, adopt two step centrifuging to prepare platelet rich plasma;
Step 3: mesenchymal stem cells MSCs-cell brick-platelet rich plasma cell conjugate preparation and injection:
Be resuspended in 500ul platelet rich plasma by Secondary Culture to the mesenchymal stem cells MSCs in P3 generation and cell brick, wherein, mesenchymal stem cells MSCs quantity is 2.25 × 10
7individual, in cell brick, initial cell quantity is 7.5 × 10
6individual;
Add in the calcium chloride solution being dissolved with 50ul thrombin after mixing, calcium chloride solution quality volume fraction is 100mg/ml, obtains cell conjugate;
This cell conjugate is transferred in 2ml syringe, after 1 minute, when cell conjugate condenses gradually, is injected in nude mice dorsal sc, through 12 weeks culturing in vivo, obtain cambium engineered cartilage.
In step one (1), film forming induced liquid is DMEM in high glucose culture medium, comprises 20% hyclone, 272ug/ml glutamine, 50ug/ml ascorbic acid, 50ug/ml penicillin, 30ug/ml streptomycin, and surplus is water.
In step one (2), alginate dissolve buffer and comprise 55mM sodium citrate, 0.15mM sodium chloride, and surplus is water, pH6.8.
In step one (2), the blade space of multi-blade is from being 1mm.
In step 2, two step centrifuging are prepared platelet rich plasma and are realized by following steps:
Whole blood room temperature 1800rpm rotating speed, after centrifugal 8 minutes, is divided into upper, middle and lower three layers, and upper strata is platelet-poor plasma, and middle level is platelet rich plasma, and lower floor is erythrocyte;
Shift upper two-layer blood plasma 3600rpm rotating speed in the centrifuge tube that another is new centrifugal 8 minutes, discard the platelet-poor plasma on upper strata 3/4, platelet is blown and beaten evenly by remaining part dropper repeatedly, is platelet rich plasma, and final platelet concentration is 20.9 ± 1.1 × 10
8/ ml, is placed in and saves backup on ice.
The present invention has the following advantages:
The present invention is on the basis of BMSCs and chondrocyte co-culture method, obtain completely biological, injectable tissue engineering bone/cartilage, general form is good, cartilage phenotype is stablized, can stable maintenance BMSCs cartilage to differentiation, hypertrophyization in effective suppression body, does not need before carrying out culturein vivo, carry out a large amount of exogenous growth factor and In vitro culture operation, solves the deficiency in background technology.
Accompanying drawing explanation
Fig. 1 is preparation flow figure of the present invention.
Fig. 2 is that cell turns cutting step figure.
Fig. 3 is cutting thickness measurement result.
Fig. 4 is the distribution situation of cell brick in PRP of B-CB-P complex.
Fig. 5 is B-CB-P, B-C-P, B-P tri-groups of sample general form observed results.
Fig. 6 be B-CB-P, B-C-P, B-P tri-groups of samples be correlated with coloration result contrast.
Fig. 7 is hypertrophyization feature base, skeletonization specific gene, later stage GAG and collagen experimental result.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention will be described in detail.
The method being obtained tissue engineering bone/cartilage by directional induction mesenchymal stem cells MSCs that the present invention relates to, is realized by following steps:
Step one: the preparation of chondrocyte and the large aggregation of extracellular matrix (cell brick):
(1) chondrocyte isolation, becomes Membrance cuiture:
That gets 1 monthly age New Zealand white rabbit enters Ear cartilage, shred rear mass fraction be 0.2% II Collagenase Type ((Gibco, the U.S.) solution is 37
zerodigest 12 hours at C temperature, afterwards, get single cell suspension in 15ml centrifuge tube with the centrifugal 5min of the rotating speed of 1000rpm;
Supernatant discarded, PBS rinses 1 time, with the centrifugal 5min of 1000rpm rotating speed, utilizes film forming induced liquid re-suspended cell, with 6.5 × 10
5individual cell/cm
2concentration plant and cultivate in 6 orifice plates, within every 3 days afterwards, change a not good liquor, culture fluid and aforementioned film forming induced liquid;
(2) chondrocyte diaphragm cell bricking process:
After 10 days, chondrocyte film forming, scrapes chondrocyte diaphragm, and it is pave in the culture dish of the sodium alginate solution of 1.2% that immersion fills mass fraction, the calcium chloride solution of instillation 102mM is until sodium alginate solution is condensed into gelatin complex completely, and thickness is 3-5mm;
Cut gelatin complex with the first horizontal rear rip cutting of multi-disc cutter, and be dipped in alginate dissolving buffer, release obtains cell brick; PBS rinses twice, for subsequent use.
In step one (1), film forming induced liquid is DMEM in high glucose culture medium (Hyclone, the U.S.), comprise 20% hyclone (Gibco, the U.S.), 272ug/ml glutamine (Amresco, the U.S.), 50ug/ml ascorbic acid (Amresco, the U.S.), 50ug/ml penicillin (Amresco, the U.S.), 30ug/ml streptomycin (Amresco, the U.S.), surplus is water.
In step one (2), alginate dissolve buffer and comprise 55mM sodium citrate (Sigma, the U.S.), and 0.15mM sodium chloride (Sigma, the U.S.), surplus is water, pH6.8.
In step one (2), multi-blade refers to the structure being fixed with multi-disc parallel blade on one piece of substrate, and see Fig. 2, blade space is from being 1mm.
Step 2: platelet rich plasma (PRP) extracts:
Get 2 monthly age New Zealand white rabbit auricular vein blood, add by 10% of cumulative volume the sodium citrate solution anticoagulant that mass fraction is 3.8%, adopt two step centrifuging to prepare platelet rich plasma.
In step 2, two step centrifuging are prepared platelet rich plasma and are realized by following steps:
Whole blood room temperature 1800rpm rotating speed, after centrifugal 8 minutes, is divided into upper, middle and lower three layers, and upper strata is platelet-poor plasma, and middle level is platelet rich plasma, and lower floor is erythrocyte;
Shift upper two-layer blood plasma 3600rpm rotating speed in the centrifuge tube that another is new centrifugal 8 minutes, discard the platelet-poor plasma on upper strata 3/4, platelet is blown and beaten evenly by remaining part dropper repeatedly, is platelet rich plasma, and final platelet concentration is 20.9 ± 1.1 × 10
8/ ml, is placed in and saves backup on ice.
Step 3: the preparation of mesenchymal stem cells MSCs-cell brick-platelet rich plasma (BMSCs-cell brick-PRP) cell conjugate and injection:
Be resuspended in 500ul platelet rich plasma by Secondary Culture to the mesenchymal stem cells MSCs in P3 generation and cell brick, wherein, mesenchymal stem cells MSCs quantity is 2.25 × 10
7individual, in cell brick, initial cell quantity is 7.5 × 10
6individual;
Add in the calcium chloride solution being dissolved with 50ul thrombin after mixing, calcium chloride solution quality volume fraction is 100mg/ml, obtains cell conjugate;
This cell conjugate is transferred in 2ml syringe, after 1 minute, when cell conjugate condenses gradually, is injected in nude mice dorsal sc, through 12 weeks culturing in vivo, obtain cambium engineered cartilage.
Hemopoietic inductive microenviroment for BMSCs cartilage to differentiation, the qualification suppressing neocartilage cellular mast and ossified effect:
After 8 weeks culturein vivos, sample is drawn materials.Carry out perusal to experimental group and matched group sample, volume, quality, thickness measure compare.Then be divided into two by each group of sample, half carries out the corresponding detection of histology, and second half carries out biochemistry and molecular Biological Detection.
1. general form observation
B-CB-P and B-C-P group sample generates white, the tough resilient tissue of matter, and B-C-P group sample is slightly harder than B-CB-P group sample.B-P group then sample generates pink, scleroid tissue.Drainage is measured each group of sample volume and is found (n=6, F=572.6, p < 0. 0001), and B-CB-P group sample volume (388.6 ± 28.6ul) is significantly greater than B-C-P(85.6 ± 6.3ul,
p< 0.01) group with B-P group (96.1 ± 8.5ul,
p< 0.01).Weight in wet base measurement result display (n=6, F=516.9,
p<0.0001) B-CB-P group specimen wet weight (356.9 ± 10.2 mg) is obviously greater than B-C-P(115.2 ± 10.9 mg,
p< 0.01) group with B-P group (49.1 ± 4.7 mg,
p< 0.01).Sample thickness (n=6, F=228.3,
p< 0. 0001) measurement result display, B-CB-P group sample thickness (3.9 ± 0.2mm) is significantly greater than B-C-P(3.0 ± 0.1mm, p < 0.01) group and B-P group (2.0 ± 0.2mm,
p< 0.01).The display of above result, cell brick and PRP jointly build volume BMSCs hemopoietic inductive microenviroment maintains cambium engineered cartilage general form stable in play a significant role, and the tissue engineering bone/cartilage of the method structure effectively maintains general form stablizes (Fig. 2).
2. stereomicroscope observation cell brick, scanning electron microscopic observation B-CB-P complex
The size utilizing stereomicroscope to measure cell brick is 1016um × 1032um, and measuring its thickness to cell brick cross-section slices is 50-80um (c1-c3 of Fig. 3).Scanning electron microscope (Jeol JSM6330F, Japan) lyophilizing 12 to be disappeared and the B-CB-P complex observed result of metal spraying process finds that cell brick is uniformly distributed in PRP, PRP fills up the space between cell brick, for BMSCs attachment, growth, differentiation provide space (b of Fig. 4).
3. histology and Immunohistochemical detection
By to dye to cell brick-PRP support HE and scanning electron microscope detects us and finds that chondrocyte turns and be uniformly distributed among PRP, form good hemopoietic inductive microenviroment, and PRP is filled between the space of cell brick formation, for BMSCs attachment, growth, differentiation provides space, and BRDU immunofluorescence dyeing proves that BMSCs is that we add, for BMSCs region safranin O and, II Collagen Type VI immunohistochemical staining proves that it is divided into neocartilage tissue.We comprise HE, safranin O dyeing by BMSCs cartilage to the relevant dyeing that differentiation is reflected, II Collagen Type VI immunohistochemical staining, find that experimental group (B-CB-P) organizes II Collagen Type VI and glycosaminoglycan (GAG) content apparently higher than matched group (Fig. 2), later stage GAG and collagen experiment further demonstrate this point (Fig. 5 E, and the ossified specific stain result of hypertrophyization (Von kossa, horse pine trichrome stain, I type, X-type collagen immunization histochemical staining) then shows that it suppresses the successful that hypertrophyization is ossify to be better than two matched groups (Fig. 3) F).Real-time quantitative reverse transcriptase polymerase chain reaction (Real time-PCR) result shows, become chondrocyte gene (II Collagen Type VI, COL-II) experimental group is higher than matched group, and hypertrophyization feature base (X-type collagen, and skeletonization specific gene (type i collagen COL-X), COL-I) experimental group is starkly lower than matched group (Fig. 5 A, B, C), these all prove that chondrocyte brick-PRP-BMSCs structure has good one-tenth cartilage characteristic and has the effect that good suppression dystopy becomes BMSCs hypertrophyization in cartilage process ossified simultaneously.
Content of the present invention is not limited to cited by embodiment, and the conversion of those of ordinary skill in the art by reading description of the present invention to any equivalence that technical solution of the present invention is taked, is claim of the present invention and contains.
Claims (5)
1. obtained the method for tissue engineering bone/cartilage by directional induction mesenchymal stem cells MSCs, it is characterized in that:
Realized by following steps:
Step one: the preparation of chondrocyte and the large aggregation of extracellular matrix:
(1) chondrocyte isolation, becomes Membrance cuiture:
That gets 1 monthly age New Zealand white rabbit enters Ear cartilage, and shredding rear mass fraction is that the II Collagenase Type solution of 0.2% is 37
zerodigest 12 hours at C temperature, afterwards, get single cell suspension in 15ml centrifuge tube with the centrifugal 5min of the rotating speed of 1000rpm;
Supernatant discarded, PBS rinses 1 time, with the centrifugal 5min of 1000rpm rotating speed, utilizes film forming induced liquid re-suspended cell, with 6.5 × 10
5individual cell/cm
2concentration plant and cultivate in 6 orifice plates, within every 3 days afterwards, change a not good liquor, culture fluid and aforementioned film forming induced liquid;
(2) chondrocyte diaphragm cell bricking process:
After 10 days, chondrocyte film forming, scrapes chondrocyte diaphragm, and it is pave in the culture dish of the sodium alginate solution of 1.2% that immersion fills mass fraction, the calcium chloride solution of instillation 102mM is until sodium alginate solution is condensed into gelatin complex completely, and thickness is 3-5mm;
Cut gelatin complex with the first horizontal rear rip cutting of multi-disc cutter, and be dipped in alginate dissolving buffer, release obtains cell brick; PBS rinses twice, for subsequent use;
Step 2: platelet rich plasma extracts:
Get 2 monthly age New Zealand white rabbit auricular vein blood, add by 10% of cumulative volume the sodium citrate solution anticoagulant that mass fraction is 3.8%, adopt two step centrifuging to prepare platelet rich plasma;
Step 3: mesenchymal stem cells MSCs-cell brick-platelet rich plasma cell conjugate preparation and injection:
Be resuspended in 500ul platelet rich plasma by Secondary Culture to the mesenchymal stem cells MSCs in P3 generation and cell brick, wherein, mesenchymal stem cells MSCs quantity is 2.25 × 10
7individual, in cell brick, initial cell quantity is 7.5 × 10
6individual;
Add in the calcium chloride solution being dissolved with 50ul thrombin after mixing, calcium chloride solution quality volume fraction is 100mg/ml, obtains cell conjugate;
This cell conjugate is transferred in 2ml syringe, after 1 minute, when cell conjugate condenses gradually, is injected in nude mice dorsal sc, through 12 weeks culturing in vivo, obtain cambium engineered cartilage.
2. the method being obtained tissue engineering bone/cartilage by directional induction mesenchymal stem cells MSCs according to claim 1, be is characterized in that:
In step one (1), film forming induced liquid is DMEM in high glucose culture medium, comprises 20% hyclone, 272ug/ml glutamine, 50ug/ml ascorbic acid, 50ug/ml penicillin, 30ug/ml streptomycin, and surplus is water.
3. the method being obtained tissue engineering bone/cartilage by directional induction mesenchymal stem cells MSCs according to claim 2, be is characterized in that:
In step one (2), alginate dissolve buffer and comprise 55mM sodium citrate, 0.15mM sodium chloride, and surplus is water, pH6.8.
4. the method being obtained tissue engineering bone/cartilage by directional induction mesenchymal stem cells MSCs according to claim 3, be is characterized in that:
In step one (2), the blade space of multi-blade is from being 1mm.
5. the method being obtained tissue engineering bone/cartilage by directional induction mesenchymal stem cells MSCs according to claim 4, be is characterized in that:
In step 2, two step centrifuging are prepared platelet rich plasma and are realized by following steps:
Whole blood room temperature 1800rpm rotating speed, after centrifugal 8 minutes, is divided into upper, middle and lower three layers, and upper strata is platelet-poor plasma, and middle level is platelet rich plasma, and lower floor is erythrocyte;
Shift upper two-layer blood plasma 3600rpm rotating speed in the centrifuge tube that another is new centrifugal 8 minutes, discard the platelet-poor plasma on upper strata 3/4, platelet is blown and beaten evenly by remaining part dropper repeatedly, is platelet rich plasma, and final platelet concentration is 20.9 ± 1.1 × 10
8/ ml, is placed in and saves backup on ice.
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| CN110172443A (en) * | 2019-05-23 | 2019-08-27 | 中国人民解放军第四军医大学 | Utilize the method for Marrow Mesenchymal Stem Cells tissue engineering bone/cartilage |
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| CN108355174A (en) * | 2018-05-06 | 2018-08-03 | 西北工业大学 | A kind of preparation method of Multifunctional layered articular cartilage holder |
| CN109517786A (en) * | 2018-10-18 | 2019-03-26 | 广州元帅生物科技有限公司 | A kind of multipotential stem cell preparation and its application with repair function |
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| CN109589451A (en) * | 2018-11-19 | 2019-04-09 | 浙江星月生物科技股份有限公司 | A kind of one-step method regenerating bone or cartilage repairing composite and preparation method thereof |
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| CN110172443A (en) * | 2019-05-23 | 2019-08-27 | 中国人民解放军第四军医大学 | Utilize the method for Marrow Mesenchymal Stem Cells tissue engineering bone/cartilage |
| CN110172443B (en) * | 2019-05-23 | 2022-07-29 | 中国人民解放军第四军医大学 | Method for constructing tissue engineering cartilage by using bone marrow mesenchymal stem cells |
| CN111514374A (en) * | 2020-04-24 | 2020-08-11 | 南京鼓楼医院 | Method for promoting cartilage repair by using tissue engineering cartilage constructed based on co-culture system |
| CN112370573A (en) * | 2020-11-04 | 2021-02-19 | 陕西佰傲干细胞再生医学有限公司 | Cartilage membrane and preparation method thereof |
| CN112370573B (en) * | 2020-11-04 | 2023-06-23 | 山东佰傲干细胞生物技术有限公司 | Cartilage diaphragm and preparation method thereof |
| CN115197906A (en) * | 2022-08-17 | 2022-10-18 | 西藏自治区人民政府驻成都办事处医院 | Method for constructing tissue engineering cartilage by using bone marrow mesenchymal stem cells |
| CN115505566A (en) * | 2022-09-30 | 2022-12-23 | 国家卫生健康委科学技术研究所 | Acellular matrix material based on human-derived mesenchymal stem cells and preparation method thereof |
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