CN103495208A - Tissue- engineered cartilage graftimplant and preparation method thereof - Google Patents
Tissue- engineered cartilage graftimplant and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a tissue tissue-engineered cartilage graftimplant and a preparation method thereof and belongs to the technical field of induced differentiation carried out on bone marrow mesenchymal stem cells (BMSCs) by utilizing a bioactive inducing factor to form a cartilage cell chondroblast composite scaffold material and so as to construct a tissue tissue-engineered cartilage by utilizing a biological activity inducing factor in biomedicine tissue engineering. The tissue tissue-engineered cartilage graftimplant is prepared by adopting the method comprising the following steps: (1) preparing a Nano-HA/PLLA (hyaluronic aciddroxyapatite/poly left L-lactic acid) cartilage scaffold material; (2) carrying out coculture on BMSCs and the Nano-HA/PLLA cartilage scaffold material, and carrying out induced differentiation on BMSCs to form cartilage cells by adopting a cartilage formation inducing solution, so that the tissue tissue-engineered cartilage graftimplant is obtained. The tissue tissue-engineered cartilage graftimplant improves flexibility and biodegradability of the cartilage scaffold material, improves biomechanical property and is more beneficial to adhesion, growth and vascularization of bone cells; an animal experiment proves that the tissue tissue-engineered cartilage graftimplant has a good cartilage defect repairing function.
Description
Technical field
The invention belongs in the biomedical tissue engineering and utilize the biological activity inducible factor to induce differentiation BMSCs chondroblast compound support frame material to build the technical field of organization engineered cartilage, relate to a kind of repair materials for cartilage defect repair, be specifically related to a kind of organization engineered cartilage graft and preparation method thereof.
Background technology
For a long time, at biomaterial and medical domain, cartilage defect repair is an important research topic always, yet clinically the reparation of the cartilage defect on a large scale that causes after wound, infection and tumor resection is not effectively solved yet up to now.Cartilage transplantation has obtained generally acknowledging of scholar as a kind of effective cartilage defect repair method, but, owing to not yet producing a kind of desirable artificial material as the cartilage transplantation substitution material at present, the research of cartilage defect repair material becomes one of hot subject in the last hundred years.Cartilage tissue engineered research mainly contains 4 aspects: timbering material, seed cell, cytokine, clinical use.Organization engineered cartilage can be avoided the defect of biogenic repair materials as the substitute of repair of cartilage material.Having of the good biocompatibility timbering material bone conduction ability and biodegradable in vivo is combined with the cytokine with powerful induced activity and can be made cartilage defect repair have bone conduction and the double grading of inducing, when rapidly becoming cartilage, embedded material is degraded gradually, for the reparation of cartilage defect provides brand-new thinking and method.
Hydroxyapatite (Hydroxy-apatite is called for short HA) is modal a kind of bioactive materials, and it has the inorganic constituents similar to body bone tissue, is the biological active ceramic material with better biocompatibility and bone conductibility of generally acknowledging at present.Polylactic acid (Polylactic acid, be called for short PLA) be current Tissue Engineering Study and the class material be most widely used, this class material non-toxic, no antigen, there is good degradable absorbability, biological safety and mechanical strength, can regulate the degradation speed of material by controlling component content, make the repeatability of product property and mechanical property reach higher level.
BMSCs mainly is present in bone marrow, now confirm that BMSCs at least can be to mature cell differentiation more than 9 kinds, comprising chondrocyte and endotheliocyte, it can become the desirable seed cell of cell therapy and organization engineered cartilage structure the prompting of differentiation polytropism, so BMSCs becomes cartilage tissue engineered desirable seed cell.
Studies have found that, during the BMSCs In vitro culture, in culture medium, adding TGF-β 1 can induce the BMSCs directed differentiation is chondrocyte, and the dosage of the expression of its II Collagen Type VI and TGF-β 1 is proportionate, and the TGF-β 1 of 5～10ng/ml can promote the differentiation of BMSCs to the cartilage direction effectively.TGF-β 1 concentration reaches 10ng/ml can make the expression of the factors such as II Collagen Type VI, Aggercan improve more than 2 times, impels BMSCs to break up to the cartilage direction.Therefore, adopt the special chondrocyte induction system of the TGF-β 1 of 10ng/ml, be expanded to the BMSCs of the third generation outside inductor to Chondrocyte Differentiation under serum-free condition, induce while within the 4th day, changing liquid first, cellular morphology expands to some extent, estimate that the possibility induced liquid infiltrates relevant, after 14 days, cell is star chondrocyte sample.
Summary of the invention
The object of the invention is to disclose a kind of organization engineered cartilage graft for cartilage defect repair.
Another object of the present invention has been to disclose its preparation method of above-mentioned organization engineered cartilage graft.
The objective of the invention is to be achieved through the following technical solutions:
A kind of organization engineered cartilage graft, comprise timbering material and seed cell, and wherein, described organization engineered cartilage graft is by following method preparation:
(1), the preparation of Nano-HA/PLLA cartilage frame material:
(a), by Nano-HA and PLLA in mass ratio 1:4 be dissolved in the organic solvent solvent, mix homogeneously, the biomaterial that obtains preparing;
(b), by the spaced parallel ice line of RIPF (Low-temperature Ice type quick shaping) process forming tool, the filling speed of RIPF technique is 20mm/s;
(c), the biomaterial prepared is inserted to the interval between the ice line, by having filled out the surface after the biomaterial, strike off, make ice line and biomaterial alternative arrangement;
(d), under orthogonal direction is shaped one deck ice line, repeating step (b) and (c);
(e), be shaped after, the entity that ice and biomaterial are formed is taken cold room, the remaining space of ice-out is tissue scaffold design required crisscross, the macropore that communicates with each other;
(f), will form porous support by biomaterial and be placed on again in freeze dryer after lyophilization, the organic solvent distillation, stayed a large amount of more fine holes;
(2), the structure of organization engineered cartilage graft:
BMSCs was passaged to for the 3rd generation by containing being inoculated in after 0.25% trypsinization of 0.02%EDTA in Nano-HA/PLLA cartilage frame material, containing in the H-DMEM culture fluid of FBS, carrying out common cultivation; When cell covers with approximately 50%～60%, by culture medium be replaced by added chondrocyte induction liquid not containing the H-DMEM culture fluid of FBS, change first culture medium after 4 days, within later every 3-4 days, change culture medium, coinduction two weeks, obtain the organization engineered cartilage graft; Described one-tenth chondrocyte induction liquid consist of TGF-β 110 μ g/L, insulin 6.25mg/L, Sodium Pyruvate 1mmol/L, vitamin C 37.5mg/L, dexamethasone 10
-7mol/L, transferrins 6.25mg/L and Monohydrated selenium dioxide 6.25mg/L.
The described organization engineered cartilage graft of technique scheme, wherein, the organic solvent in step (a) is the 1-4 dioxane; Tissue scaffold design in step (e) is three-dimensional porous structure, and hole is 200-500 μ m, and porosity is 80%-90%, and hole is interconnected pore.
The described organization engineered cartilage graft of technique scheme, wherein, the inoculum density that BMSCs is inoculated in Nano-HA/PLLA cartilage frame material after trypsinization is 6 * 10
3/ cm
2.
The preparation method of the described organization engineered cartilage graft of technique scheme, comprise the steps:
(1), the preparation of Nano-HA/PLLA cartilage frame material:
(a), by Nano-HA and PLLA in mass ratio 1:4 be dissolved in the organic solvent solvent, mix homogeneously, the biomaterial that obtains preparing;
(b), by the spaced parallel ice line of RIPF process forming tool, the filling speed of RIPF technique is 20mm/s;
(c), the biomaterial prepared is inserted to the interval between the ice line, by having filled out the surface after the biomaterial, strike off, make ice line and biomaterial alternative arrangement;
(d), under orthogonal direction is shaped one deck ice line, repeating step (b) and (c);
(e), be shaped after, the entity that ice and biomaterial are formed is taken cold room, the remaining space of ice-out is tissue scaffold design required crisscross, the macropore that communicates with each other;
(f), will form porous support by biomaterial and be placed on again in freeze dryer after lyophilization, the organic solvent distillation, stayed a large amount of more fine holes;
(2), the structure of organization engineered cartilage graft:
BMSCs was passaged to for the 3rd generation by containing being inoculated in after 0.25% trypsinization of 0.02%EDTA in Nano-HA/PLLA cartilage frame material, containing in the H-DMEM culture fluid of FBS, carrying out common cultivation; When cell covers with approximately 50%～60%, by culture medium be replaced by added chondrocyte induction liquid not containing the H-DMEM culture fluid of FBS, change first culture medium after 4 days, within later every 3-4 days, change culture medium, coinduction two weeks, obtain the organization engineered cartilage graft; Described one-tenth chondrocyte induction liquid consist of TGF-β 110 μ g/L, insulin 6.25mg/L, Sodium Pyruvate 1mmol/L, vitamin C 37.5mg/L, dexamethasone 10
-7mol/L, transferrins 6.25mg/L and Monohydrated selenium dioxide 6.25mg/L.
The preparation method of the described organization engineered cartilage graft of technique scheme, wherein, the organic solvent in step (a) is the 1-4 dioxane; Tissue scaffold design in step (e) is three-dimensional porous structure, and hole is 200-500 μ m, and porosity is 80%-90%, and hole is interconnected pore.
The preparation method of the described organization engineered cartilage graft of technique scheme, wherein, the inoculum density that BMSCs is inoculated in Nano-HA/PLLA cartilage frame material after trypsinization is 6 * 10
3/ cm
2.
The present invention has following beneficial effect:
1, the present invention utilizes RIPF (low temperature rapid shaping) technology by Nano-HA and the three-dimensional porous Nano-HA/PLLA artificial bone of the compound preparation of PLLA, this three-dimensional porous artificial bone supporting material combines the advantage of Nano-HA and PLLA, make it on the basis of good bone conductibility and biocompatibility, improve pliability and biological degradability, improve biomechanical property, more be conducive to adhesion growth and the vascularization of osteocyte, the damaged place of bone be will greatly improve and artificial symphysis speed and effect transplanted, it is a kind of desirable novel nano combined artificial bone holder material.
2, the present invention adopts density gradient centrifugation associating Attachment culture BMSCs, has greatly improved the success rate of separating.And in culture medium, add inducible factor TGF-β 1 to Chondrocyte Differentiation, can bring into play to greatest extent the action time of TGF-β 1.
3, the organization engineered cartilage graft of structure of the present invention can be for repairing the cartilage graft of cartilage defect, verifiedly in zoopery can well repair cartilage defect.
The accompanying drawing explanation:
1, Fig. 1 is Nano-HA/PLLA cartilage frame material microstructure observed result (SEM50 *) in the embodiment of the present invention 3;
2, observed result (SEM5000 *) under the organization engineered cartilage graft scanning electron microscope that Fig. 2 is preparation in the embodiment of the present invention 3;
3, Fig. 3 is for inducing rear chondrocyte II Collagen Type VI immunohistochemical staining positive expression;
4, Fig. 4 is the expression that RT-PCR detects compound rear inducing cell Aggrecan, col2A1;
5, Fig. 5 is the expression that Western-bolt detects compound rear inducing cell II collagen type;
6, Fig. 6 is gross examination of skeletal muscle figure when in the embodiment of the present invention 4, A organizes 24 weeks;
7, Fig. 7 is gross examination of skeletal muscle figure when in the embodiment of the present invention 4, B organizes 24 weeks;
8, Fig. 8 is gross examination of skeletal muscle figure when in the embodiment of the present invention 4, C organizes 24 weeks;
9, Fig. 9 is histology figure when in the embodiment of the present invention 4, A organizes 24 weeks;
10, Figure 10 is histology figure when in the embodiment of the present invention 4, B organizes 24 weeks;
11, Figure 11 is histology figure when in the embodiment of the present invention 4, C organizes 24 weeks.
The specific embodiment:
For making technical scheme of the present invention be convenient to understand, below in conjunction with concrete test example, a kind of organization engineered cartilage graft of the present invention and preparation method thereof is further described.
embodiment 1:the preparation of Nano-HA:
Adopt colloidal sol-flocculence synthesizing hydroxylapatite.Preparation method: the aqueous solution of lime nitrate and ammonium phosphate is carried out to chemosynthesis, add a certain amount of ammonia in building-up process, the pH value of adjusting solution is 8～13, add dispersant, select suitable agitator, adjust agitator speed and mixing time, make its precipitation fully, then through washing, filtration, by precipitate 80～120 ℃ of dryings, at 600～800 ℃ of temperature, sintering is 2～3 hours, by obtaining powder diameter, is less than the 100nm nanometer hydroxyapatite powder similar to the body bone tissue composition.
1, the preparation of lactide: extracting lactic acid and ZnO powder, heat up and make acid by dehydrating lactic become lactide gradually; Then the evacuation that heats up steams faint yellow lactide monomer, then take ethyl acetate as solvent cyclic washing, recrystallization purification, sucking filtration, and vacuum drying, obtain the pure lactide crystal of colourless lamellar.
2, PLLA's is synthetic: pure lactide monomer, add the stannous octoate initiator, under room temperature after the vacuum drying certain hour, soak 3-5min in silicone oil bath, the sealed reaction bottle, be warming up to when monomer has just melted and repeatedly shake reaction bulb to mix homogeneously, then clock and be polymerized to certain hour taking-up reaction bulb.
3, the purification of PLLA: in rough PLLA, add dichloromethane that it is dissolved fully, filter, in filtrate, add methanol complete to precipitation, refilter, methanol wash, vacuum drying, obtain white pure PLLA.
4, the mensuration of PLLA molecular weight: take a certain amount of PLLA and be dissolved in chloroform, measure with the Ubbelohde viscosimetry, calculate relatively sticky equal molecular mass.
Raw material Nano-HA in the following embodiment of the present invention and PLLA can buy and also can be prepared by the method for embodiment 1 and 2 from market.
embodiment 3:the preparation of organization engineered cartilage graft:
A kind of organization engineered cartilage graft comprises timbering material and adheres to the seed cell on timbering material that this seed cell is to be induced the chondrocyte be divided into through TGF-β 1 by BMSCs; The preparation method of this organization engineered cartilage graft is as follows:
1, the preparation of Nano-HA/PLLA cartilage frame material
(1), the preparation of Nano-HA/PLLA cartilage frame material:
(a), by Nano-HA and PLLA in mass ratio 1:4 be dissolved in the organic solvent solvent, mix homogeneously, the biomaterial that obtains preparing;
(b), by the spaced parallel ice line of RIPF process forming tool, the filling speed of RIPF technique is 20mm/s, the speed of filling has determined the gap size between parallel ice line;
(c), the biomaterial prepared is inserted to the interval between the ice line, by having filled out the surface after the biomaterial, strike off, make ice line and biomaterial alternative arrangement;
(d), under orthogonal direction is shaped one deck ice line, repeating step (b) and (c);
(e), be shaped after, the entity that ice and biomaterial are formed is taken cold room, the remaining space of ice-out is tissue scaffold design required crisscross, the macropore that communicates with each other;
(f), will form porous support by biomaterial is placed in freeze dryer after lyophilization again, the organic solvent distillation, a large amount of more fine holes have been stayed, obtain Nano-HA/PLLA cartilage frame material, as shown in Figure 1, wherein Fig. 1 is that Nano-HA/PLLA cartilage frame material microstructure is observed (SEM50 *).
2, BMSCs preparation:
Get the new zealand white rabbit of 2-3 monthly age, body weight 2.0-2.5kg, adopt that density－gradient centrifuga－tion method associating adherent method separates, the L-DMEM culture fluid of 10%FBS is cultivated rBMSCs, when primitive cell culture after 1 week, with 1:2 or 1:1 had digestive transfer culture, within 2-3 days, change liquid 1 time, be passaged to after the 3rd generation standby.
3, the preparation of organization engineered cartilage graft:
After BMSCs was passaged to for the 3rd generation, with 0.25% trypsin that contains 0.02%EDTA, digested, then with 6 * 10
3/ cm
2density be inoculated in Nano-HA/PLLA cartilage frame material, carry out common cultivation in aseptic 6 well culture plates that are equipped with containing the H-DMEM culture fluid of FBS;
When cell covers with approximately 50%～60% when the ratio of adherent covering (cell grow in culture dish), culture medium is replaced by the H-DMEM culture fluid that does not contain FBS, containing the H-DMEM culture fluid of FBS, do not adding into chondrocyte induction liquid, what become chondrocyte induction liquid consists of TGF-β 110 μ g/L, insulin 6.25mg/L, Sodium Pyruvate 1mmol/L, vitamin C 37.5mg/L, dexamethasone 10
-7mol/L, transferrins 6.25mg/L and Monohydrated selenium dioxide 6.25mg/L; Change first liquid after 4 days, within later every 3-4 days, change induced liquid, coinduction two weeks, obtain the organization engineered cartilage graft, as shown in Figure 2, wherein Fig. 2 observes (SEM5000 *) under organization engineered cartilage graft (being that BMSCs induces the composite three-dimensional porous Nano-HA/PLLA artificial cartilage of differentiating cartilage-forming cell timbering material through TGF-β 1) scanning electron microscope, and electron-microscope scanning is shown in that noble cells is evenly distributed at timbering material, sticks good.
BMSCs induces backward Chondrocyte Differentiation through TGF-β 1, break up as seen chondrocyte secretion glycosaminoglycans (glycosaminoglycan through Toluidine blue staining, GAG), II Collagen Type VI immunohistochemical staining be positive (see Fig. 3, Fig. 3 is for inducing rear chondrocyte II Collagen Type VI immunohistochemical staining positive expression);
4, RT-PCR detects the expression of differentiation chondrocyte related gene in composite:
Collect and attach the support cell of the 7th, 14,21 days and extract total RNA according to Trizol (Invitrogen, USA) reagent description operation digestion.According to the test kit operation of Fermentas company, the total RNA reverse transcription of 2ul is become to cDNA, system is 40ul.
PCR instrument (Eppendorf) amplifying target genes, contain 2 * PCR Master mix25ul in 50 μ l PCR reaction systems, cDNA3 μ l, each 2 μ l of upstream and downstream primer, primer sequence and product length are in Table 1, and primer is insulted the bio tech ltd design by glad sea, Shenzhen.
Amplification condition is 94 ℃ of denaturation 5min, 94 ℃ of 30s, and 56 ℃ of 30s, 72 ℃ of 45s, totally 30 circulations, 72 ℃ of 10min extend, and using the GAPDH gene as endogenous reference gene (table 1).
Table 1PCR primer series and product length
The results are shown in Figure shown in 4, Fig. 4 is the expression that RT-PCR detects compound rear inducing cell Aggrecan, col2A1; Wherein row 1 are that 7 days, row 2 are that 14 days, row 3 are 21 days.
5, Western Blot detects differentiation chondrocyte II collagen type (Col2) secretion situation in composite:
After getting respectively the 7th, 14,21 days cell carrier complex (organization engineered cartilage graft) PBS washing three times, 0.25% trypsinization 2min, add containing the 10%FBS culture fluid and stop digestion.After piping and druming 3min, cell suspension is transferred to centrifuge tube 1500rpm, centrifugal 3min, abandon and reset and add Trizol extraction peptic cell total protein, usings GAPDH as positive control; 10%SDS-PAGE gel electrophoresis (Biorad vertical electrophoresis apparatus), 10 μ l loadings, 110V, electrophoresis 60min; 15V voltage transfer printing 70min (the half-dried transferring film instrument of Biorad SD type) afterwards; The anti-rabbit antibody of II Collagen Type VI monoclonal antibody (primary antibodie) 5 μ l add 1000 times of 10%TBST5ml dilutions, and 4 ℃ of sealings are spent the night; 10%TBST washs 3 times (5min time), and 1:1000 mouse-anti rabbit igg (two is anti-) is hatched 3 times (5min time) of 10%TBST washing after hour, and ECL develops the color and scans and carries out quantitative analysis.
The results are shown in Figure shown in 5, Fig. 5 is the expression that Western-bolt detects compound rear inducing cell II collagen type; Wherein row 1 are that 7 days, row 2 are that 14 days, row 3 are 21 days.
RT-PCR and Western-bolt detect and show that 7,14,21 days Aggrecan, Col2A1 all have in various degree and express at mRNA level, II collagen type.
The present invention's Nano-HA/PLLA cartilage frame material used is the three-dimensional porous nano compound support frame material, recording hole by the SEM electron-microscope scanning is 200-500 μ m, porosity is 80%-90%, and the gained hole is interconnected pore, compare and there is better mechanical strength with single biomaterial, the biodegradability be more suitable for, biocompatibility preferably.
The testing procedure of porosity is as follows: by weight, be W
sporous support put into the 100ml round-bottomed flask, after evacuation, with syringe, by the ethanol injection bottle, logical atmosphere, make ethanol enter internal stent under the effect of pressure fast; By evacuation several times/logical Atmospheric processes, the hole in support is taken fully by ethanol; This support is put into to a weighing botle that has filled with in advance ethanol, and W weighs
1.After support is carefully taken out from bottle, the weight W of the weighing botle of again weighing
2.The porosity of support is calculated according to following formula:
ρ in formula
edensity for timbering material
V
svolume for ethanol
embodiment 4:the application of organization engineered cartilage graft in the animal cartilage defect repair:
One, zoopery operation technique:
1, animal grouping:
18 new zealand white rabbits, body weight (2.5-3.0kg), male and female are not limit, and each knee joint, as a unit, is divided 3 groups by 18 rabbits, every group of 6 rabbits, 12 joints.Experimental group (A group): the organization engineered cartilage graft (embodiment 3 preparations) of cultivating 2 weeks is transplanted at damaged place; Simple support group (B group): the aseptic timbering material of prewetting is transplanted at damaged place; Blank group (C group): damaged place does not process.Respectively at postoperative 24 weeks every group put to death all experimental rabbits and draw materials, observe the situation that repair at damaged place.
2, operation method:
18 of new zealand white rabbits, first feed and conform in 1 week.Intramuscular injection speed is slept new (0.3ml/kg), in conjunction with chlore-ammonia ketone 0.1g/kg intraperitoneal injection of anesthesia, and after anaesthetizing successfully, drape, the inboard patella escribe of the descending knee joint of aseptic condition mouth, stir patella laterally, appears lateral condyle and coaster in femur.At condylus medialis femoris with special Twist Drill Hole, trepan overall diameter 4.5mm, drilling through the degree of depth is 5mm, causes cylindrical cartilage and subchondral bone defective region.
According to random packet, the A group is at damaged place transplanted tissue through engineering approaches cartilage graft; The B group is transplanted aseptic support at damaged place; C organizes spacious the putting in damaged place and does not process.Clean wound, sew up joint capsule and skin.Postoperative every rabbit intramuscular injection 400,000 unit penicillins, for three days on end, in case knee joint infects.Postoperative nursing as usual, the art limb is without braking.
Two, observation index and method:
1, postoperative ordinary circumstance:
The postoperative limb activity of animal and diet situation, wound has or not infection.
2, gross examination of skeletal muscle:
24 weeks dead 6 rabbits in every component other places, the gross examination of skeletal muscle rabbit knee had or not swelling, adhesion, hydrops, shape, color and luster, the smoothness that repair at damaged place, have or not depression, and the surrounding normal cartilage in conjunction with situation.
3, histological observation:
Take off condyle in rabbit femoral and comprise the reparation specimen of art district and surrounding normal cartilage and subchondral bone, 10% formaldehyde is fixed 1 week, 5% salpeter solution decalcification 4-5 days is to the specimen deliquescing, flowing water rinses, gradient alcohol dehydration: by 70% ethanol 1h, 80% ethanol 1h, 90% ethanol 1h, 95% ethanol 1h, 100% ethanol I 30min, 100% ethanol II 30min, dimethylbenzene is transparent: dimethylbenzene I 30min, dimethylbenzene II 30min, 60 ℃ of lower waxdip 4h, paraffin embedding, with 5 μ serial section, section is put into to 45 ℃ of warm water and flatten, microscope slide is dried, and prepares dyeing.
3.1, HE dyeing:
Get above-mentioned section and be placed in the roasting sheet 30min of 60 ℃ of constant temperature roasters, dimethylbenzene and gradient ethanol dewaxing aquation, order is: dimethylbenzene I 15min, dimethylbenzene II 15min, dehydrated alcohol I 2min, dehydrated alcohol II 2min, 95% ethanol 2min, 80% ethanol 2min, 70% ethanol 2min, flowing water 2min, distilled water 2min.The haematoxylin dye liquor dyes core 5min, and mobile tap water expresses, 1% acidic alcohol (70%) the differentiation several seconds, wash from the beginning 30min, " oil blackeite " 3-4 second in ammonia, rinsing 5min (changing once) in distilled water, microscopy, differentiation degree becomes blue with karyon, background approaches colourless for good, as breaks up excessively, can return and dye, as break up deficiency, can again break up.Then enter eosin stain dyeing 1-5min.Gradient ethanol dehydration: 70% ethanol several seconds, 80% ethanol 2min, 95% ethanol 2min, dehydrated alcohol I 2min, dehydrated alcohol II 2min, dehydrated alcohol and dimethylbenzene equivalent mixed liquor 5min, dimethylbenzene is transparent: dimethylbenzene 5-10min, the neutral gum mounting, under inverted microscope, observe, it is black-and-blue that nucleus is, and Cytoplasm is red.
3.2, the fast green dyeing of kind O/:
Get above-mentioned section and be placed in the roasting sheet 30min of 60 ℃ of calorstats, dimethylbenzene and gradient ethanol dewaxing aquation: dimethylbenzene I 15min, dimethylbenzene II 15min, dehydrated alcohol I 2min, dehydrated alcohol II 2min, 95% ethanol 2min, 80% ethanol 2min, 70% ethanol 2min, flowing water 2min, distilled water 2min.2% fast green water dyes 3min, 1% safranin O dyeing 3min, and the distillation washing, the gradient ethanol dehydration, dimethylbenzene is transparent, and the neutral gum sealing is observed under inverted microscope.
3.3, II Collagen Type VI immunohistochemical staining:
Mouse-anti rabbit II Collagen Type VI monoclonal antibody (1:50) for primary antibodie, two is anti-with the anti-Mus two of biotinylation rabbit anti-(1:80), the DAB colour developing.Concrete grammar following (pressing the S-P method).Get above-mentioned section and be placed in the roasting sheet 30min of 60 ℃ of calorstats, dimethylbenzene and gradient ethanol dewaxing aquation: dimethylbenzene I 15min, dimethylbenzene II 15min, dehydrated alcohol I 2min, dehydrated alcohol II 2min, 95% ethanol 2min, 80% ethanol 2min, 70% ethanol 2min, flowing water 2min, distilled water 2min.At first drip 50 μ l peroxidase blocking solutions (reagent A), incubated at room 15min, with the activity of blocking-up endogenous peroxydase, PBS cleans 3 times (3min/ time); Add 1 nonimmune animal serum (reagent B) after sucking-off PBS liquid, incubated at room 15min; PBS adds the first antibody of 50 μ l after cleaning, incubated at room 80min, and PBS rinses three times; Go to add the biotin labeled second antibody of 50 μ l after PBS liquid, under room temperature, hatch 10min; PBS adds 50 μ l streptomycete antibiotins-peroxide enzymatic solution (reagent D) after rinsing, and under room temperature, hatches 15min; PBS adds 2 DAB solution after rinsing, and after microscopic examination, tap water rinses, and haematoxylin is redyed, and tap water rinses and returns indigo plant, the DAB colour developing; Section is through the gradient alcohol dehydration drying, and dimethylbenzene is transparent, the neutral gum sealing.
Three, result:
1, ordinary circumstance:
After anesthesia is clear-headed, animal activity is few, diet reduces, and after 2-3 days, gait and diet recover, and in nursing, redness does not appear in wound, and wound healing is good.
2, gross examination of skeletal muscle:
(1), the result of 12 weeks: gross examination of skeletal muscle shows that A group defective region is filled, and surface is slightly smooth, and edge is integrated; B organizes slight depression, air spots, visible support; C group depression is dark, can see subchondral bone.
(2), the result of 24 weeks: A group defect surfaces becomes smooth, smooth, and edge is integrated, as shown in Figure 6; B group depression shoals, but surface is still rough and uneven in surface, and edge is integrated still undesirable, as shown in Figure 7; The result of C group depression than 12 weeks shoals, and Defect diameter diminishes, but central authorities' visible subchondral bone still, as shown in Figure 8.
3, histology:
The A group:
(1), the sample of 12 weeks shows normal or slight ultra-fine born of the same parents' structure, compare the band shape that normal histiocytic arrangement lacks typical chondrocyte and arrange.Fibrous tissue and porous support are occupied an leading position in damaged, but the tissue of class cartilage feature occurs on damaged surface.Kind O stained shows that whole graft area has sGAG.The immunostaining of II Collagen Type VI shows: typeⅡ Collagen is the different chondrocyte secretions of whole graft area.Except the tissue of the whole reparation in surface is colored, there is intensive dyeing district part.
(2), 24 weeks graft area, at the defective region of near surface thoroughly by the chondrocyte tissue filling, the subchondral bone of bottom formation rule.The cartilage of tighter integration and bone.Compare with normal cartilage, the cartilage layers of calcification is arranged at interface formation one diatom.Yet in some samples, the formation of subchondral bone is incomplete, causes the part boundary.And, in some situation, repair tissue and normal structure integration place have crack and crackle, without the cartilaginous tissue place, the same distribution of kind O dyeing and immunostaining demonstration sGAG and typeⅡ Collagen, similar with normal cartilage, as shown in Figure 9.In all samples, the closeness of transplanting place typeⅡ Collagen is lower than the normal structure, shows in rebuilding tissue the amount of typeⅡ Collagen or few than normal articular cartilage.
The B group:
(1), the transplantation group of 12 weeks has relatively smooth surface fiber tissue to form, the Nano-HA/PLLA loose structure is still visible, is assembling circular cell around especially in hole.In addition, there is no the typeⅡ Collagen deposition so both contained large stretch of cartilaginous tissue of sGAG in section yet.
And some situation (2), although 24 weeks transplantation group are damaged smooth surface arranged, do not have cartilage layers to form, kind O is no dyeing also, shows and do not have chondrocyte to form, as shown in figure 10.
The C group:
(1), the sample of 12 weeks has the surface of irregular and fiber, to sGAG and the inhomogeneous dyeing of typeⅡ Collagen.
(2), the sample of 24 weeks shows, fiber filaments accounts for leading in damaged, damagedly fills fully and does not have cartilage layers to form simultaneously.After dyeing, the whole graft area of electron-microscope scanning does not almost have sGAG and typeⅡ Collagen, as shown in figure 11.
The above, it is only preferred embodiment of the present invention, not the present invention is done to any formal and substantial restriction, all those skilled in the art, within not breaking away from the technical solution of the present invention scope, when utilizing the disclosed above technology contents, and the equivalent variations of a little change of making, modification and differentiation is equivalent embodiment of the present invention; Simultaneously, the change of any equivalent variations that all foundations essence technology of the present invention is done above embodiment, modification and differentiation, all still belong in the scope of technical scheme of the present invention.
Claims (6)
1. an organization engineered cartilage graft, comprise timbering material and seed cell, it is characterized in that, described organization engineered cartilage graft is by following method preparation:
(1), the preparation of Nano-HA/PLLA cartilage frame material:
(a), by Nano-HA and PLLA in mass ratio 1:4 jointly be dissolved in the organic solvent solvent, mix homogeneously, the biomaterial that obtains preparing;
(b), by the spaced parallel ice line of RIPF process forming tool, the filling speed of RIPF technique is 20mm/s;
(c), the biomaterial prepared is inserted to the interval between the ice line, by having filled out the surface after the biomaterial, strike off, make ice line and biomaterial alternative arrangement;
(d), under orthogonal direction is shaped one deck ice line, repeating step (b) and (c);
(e), be shaped after, the entity that ice and biomaterial are formed is taken cold room, the remaining space of ice-out is tissue scaffold design required crisscross, the macropore that communicates with each other;
(f), will form porous support by biomaterial and be placed on again in freeze dryer after lyophilization, obtain Nano-HA/PLLA cartilage frame material;
(2), the structure of organization engineered cartilage graft:
BMSCs was passaged to for the 3rd generation by containing being inoculated in after 0.25% trypsinization of 0.02%EDTA in Nano-HA/PLLA cartilage frame material, containing in the H-DMEM culture fluid of FBS, carrying out common cultivation; When cell covers with approximately 50%～60%, by culture medium be replaced by added chondrocyte induction liquid not containing the H-DMEM culture fluid of FBS, change first culture medium after 4 days, within later every 3-4 days, change culture medium, coinduction two weeks, obtain the organization engineered cartilage graft; Described one-tenth chondrocyte induction liquid consist of TGF-β 110 μ g/L, insulin 6.25mg/L, Sodium Pyruvate 1mmol/L, vitamin C 37.5mg/L, dexamethasone 10
-7mol/L, transferrins 6.25mg/L and Monohydrated selenium dioxide 6.25mg/L.
2. according to the described organization engineered cartilage graft of right 1, it is characterized in that: the organic solvent in step (a) is the 1-4 dioxane; Tissue scaffold design in step (e) is three-dimensional porous structure, and hole is 200-500 μ m, and porosity is 80%-90%, and hole is interconnected pore.
3. according to the described organization engineered cartilage graft of right 1, it is characterized in that: the inoculum density that BMSCs is inoculated in Nano-HA/PLLA cartilage frame material after trypsinization is 6 * 10
3/ cm
2.
4. the preparation method of organization engineered cartilage graft claimed in claim 1, comprise the steps:
(1), the preparation of Nano-HA/PLLA cartilage frame material:
(a), by Nano-HA and PLLA in mass ratio 1:4 be dissolved in the organic solvent solvent, mix homogeneously, the biomaterial that obtains preparing;
(b), by the spaced parallel ice line of RIPF process forming tool, the filling speed of RIPF technique is 20mm/s;
(c), the biomaterial prepared is inserted to the interval between the ice line, by having filled out the surface after the biomaterial, strike off, make ice line and biomaterial alternative arrangement;
(d), under orthogonal direction is shaped one deck ice line, repeating step (b) and (c);
(e), be shaped after, the entity that ice and biomaterial are formed is taken cold room, the remaining space of ice-out is tissue scaffold design required crisscross, the macropore that communicates with each other;
(f), will form porous support by biomaterial and be placed on again in freeze dryer after lyophilization, obtain Nano-HA/PLLA cartilage frame material;
(2), the structure of organization engineered cartilage graft:
BMSCs was passaged to for the 3rd generation by containing being inoculated in after 0.25% trypsinization of 0.02%EDTA in Nano-HA/PLLA cartilage frame material, containing in the H-DMEM culture fluid of FBS, carrying out common cultivation; When cell covers with approximately 50%～60%, by culture medium be replaced by added chondrocyte induction liquid not containing the H-DMEM culture fluid of FBS, change first culture medium after 4 days, within later every 3-4 days, change culture medium, coinduction two weeks, obtain the organization engineered cartilage graft; Described one-tenth chondrocyte induction liquid consist of TGF-β 110 μ g/L, insulin 6.25mg/L, Sodium Pyruvate 1mmol/L, vitamin C 37.5mg/L, dexamethasone 10-7mol/L, transferrins 6.25mg/L and Monohydrated selenium dioxide 6.25mg/L.
5. preparation method according to claim 4, it is characterized in that: the organic solvent in step (a) is the 1-4 dioxane; Tissue scaffold design in step (e) is three-dimensional porous structure, and hole is 200-500 μ m, and porosity is 80%-90%, and hole is interconnected pore.
6. preparation method according to claim 4, it is characterized in that: the inoculum density that BMSCs is inoculated in Nano-HA/PLLA cartilage frame material after trypsinization is 6 * 10
3/ cm
2.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105343936A (en) * | 2015-11-05 | 2016-02-24 | 深圳市第二人民医院 | Poly-L-lactide-caprolactone copolymer (PLCL) three-dimensional porous scaffold, PLCL and collagen (PLCL-COL) composite scaffold and preparation methods of scaffolds |
| CN106434538A (en) * | 2016-09-29 | 2017-02-22 | 广州赛莱拉干细胞科技股份有限公司 | Three-dimensional chondrocyte culture method, made chondrocyte and application |
| CN109316632A (en) * | 2018-11-15 | 2019-02-12 | 北京大学口腔医学院 | A kind of preparation method of left-handed hydrogel material |
| WO2020156388A1 (en) * | 2019-01-31 | 2020-08-06 | 华东理工大学 | New use of stem cell generator in preparation of bone defect repair materials |
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2013
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| 吴任东等: "低温冰型在骨组织支架成形中的应用", 《低温工程》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105343936A (en) * | 2015-11-05 | 2016-02-24 | 深圳市第二人民医院 | Poly-L-lactide-caprolactone copolymer (PLCL) three-dimensional porous scaffold, PLCL and collagen (PLCL-COL) composite scaffold and preparation methods of scaffolds |
| CN106434538A (en) * | 2016-09-29 | 2017-02-22 | 广州赛莱拉干细胞科技股份有限公司 | Three-dimensional chondrocyte culture method, made chondrocyte and application |
| CN109316632A (en) * | 2018-11-15 | 2019-02-12 | 北京大学口腔医学院 | A kind of preparation method of left-handed hydrogel material |
| WO2020156388A1 (en) * | 2019-01-31 | 2020-08-06 | 华东理工大学 | New use of stem cell generator in preparation of bone defect repair materials |
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