CN103638558B - In vitro construction method for bionic ligament-bone tissue engineering connector - Google Patents
In vitro construction method for bionic ligament-bone tissue engineering connector Download PDFInfo
- Publication number
- CN103638558B CN103638558B CN201310462274.8A CN201310462274A CN103638558B CN 103638558 B CN103638558 B CN 103638558B CN 201310462274 A CN201310462274 A CN 201310462274A CN 103638558 B CN103638558 B CN 103638558B
- Authority
- CN
- China
- Prior art keywords
- cell
- section
- preparation
- heel string
- chondrocyte
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the interface tissue engineering technology field, and relates to an in vitro construction method for a bionic ligament-bone connector. A new choice is provided for reconstruction of a continuous ligament-bone connector, with heterogeneous gradient structures between ligament-bone interfaces after ACL damage. The technical scheme is an in vitro construction method for a bionic ligament-bone connector. The method comprises the following steps: first, preparation of acellular tendo calcaneus is carried out; second, preparation of seed cells is carried out, and the preparation comprises preparation of fibrocytes, chondrocytes and osteoblasts; third, implantation is carried out. The method provides a new strategy for tissue engineering regeneration of anterior cruciate ligament damage.
Description
Technical field
The invention belongs to Interface Microstructure field of engineering technology, relate to the vitro construction method of a kind of bionics ligament-bone connector.
Background technology
Anterior cruciate ligament (anterior cruciate ligament, ACL) maintains knee joint to stablize the important feature with function.But, cell distribution and blood for difference anatomical features determine cruciate ligament after be difficult to spontaneous recovery, cause the complication such as joint instability, cartilage injury, osteoarthritis, have a strong impact on knee joint function.Epidemiological study has been carried out in the ligamentous injury of knee joint to their national in January, 2000 in June, 2005 such as Simon M, the knee ligament operation of result display 80% is relevant with the damage of ACL, and the damage mechanisms of ACL probably can divide for the indirect injury caused by the stress that turns up and the noncontact caused by such as bone injury to damage two kinds.
The conventional main flow therapeutic strategy of ACL damage be autologous bone-kneecap tendon-Gu with hamstring tendon transplantation, but owing to taking from body tissue injury (or sacrifice) original bone-kneecap tendon-Gu with hamstring tendon.The allograft treatments ACL that adopts damages more in recent years, but its source is limited and with high costs, and late result is also undesirable simultaneously, and mortality is 10%-25%.Find when failure case is overhauled that ， Hams is rebuild structure and is partial to fiber characteristics, and patellar ligament reconstruction structure is partial to cartilage feature.Structure after failed reason is summed up as reconstruction more does not have the poor healing that between normal ACL-position of bone point fibrous cartilage, good blood supply causes; Or relax and even fracture for " rain brush effect " makes osseous tunnel expansion, graft wearing and tearing and then cause.Research is separately had to observe the histological change at tendon-osseous tunnel interface after Reconstruction: 2 weeks visible scar tissue filling gaps, dense connective tissue is developed into after January, after 6 weeks, visible type i collagen distributes in a large number, but does not find the formation of obvious area of new bone and cartilage yet to March.
Research finds: between normal ligament-osseous tissue 100 μm of-1mm thickness space in there is a transitional zone (continuous gradients layers) that is complicated, continuous distribution, or title transition complex (heterogeneous transition complex): structure from shallow to deep and corresponding composition thereof are followed successively by: fibrous layer (fibroblast and type i collagen), non-calcified fibrous cartilage (oval chondrocyte and II Collagen Type VI), calcification fibrous cartilage (hypertrophic chondrocyte and X-type collagen), subchondral bone.Namely the Gradient distribution of heterogeneous cell, extracellular matrix, mineralization degree is shown as, this buffer structure can make the complex load between dissimilar tissue and strain redistribution, dispersing shear, avoid stress to concentrate, and ensures that the dissection of ligament-subchondral bone connection is complete and stablizes with mechanics.If do not have the fibrous cartilage transition region of structure gradual change to be formed and suitable organizational integration between the research such as Kurosaka M display surgical reconstruction graft and osseous tunnel, the mechanical stability of anterior cruciate ligament graft will be greatly affected.
Discuss by above, the biological interest that cross ligament damage is rebuild should be continuous print between ligament-bone interface, there is heterogeneity to divide a word with a hyphen at the end of a line the ligament-bone connector of structure.
Summary of the invention
The technical problem to be solved in the present invention is for continuous print between ligament-bone interface after ACL damage, has the divide a word with a hyphen at the end of a line ligament-bone connector of structure of heterogeneity and provide a kind of and newly to select.
Technical scheme of the present invention is the vitro construction method of a kind of bionics ligament-bone connector, comprises the steps:
The preparation of a, de-cell heel string;
The preparation of b, seed cell: comprise fibrocyte, the chondrocyte of Sox-9 genes amplification, the osteoblastic preparation of Runx-2 genes amplification and gene transfection;
C, plantation: de-cell heel string is divided into into segment of fiber FB-according to its longitudinal axis successively with transverse axis trend and becomes cartilage section CH-skeletonization section OS tri-26S Proteasome Structure and Function regions, the chondrocyte layering plantation of the osteoblast of fibrocyte, Runx-2 genes amplification, Sox-9 genes amplification, segmentation are planted into segment of fiber, become cartilage section, skeletonization section, after cultivating histology's checking in 5 ~ 7 days, then cultivate use in 1 week; Described layering plantation refers to is transplanted to center, transverse section by heel string surface, makes the osteoblast of fibrocyte, Runx-2 genes amplification, interior, domestic and abroad each 1/3 region that the chondrocyte of Sox-9 genes amplification lays respectively at transverse section.
Wherein, in step a, de-cell heel string support is the heel string tissue deriving from rabbit, dog, cattle.
Wherein, in step b, osteoblastic preparation comprises the steps: the adenovirus vector of construction expression early stage skeletonization mark RUNX-2, transfection osteoblast.
Wherein, in step b, the preparation of chondrocyte comprises the steps: the adenovirus vector of construction expression early stage cartilage mark SOX-9, transfection chondrocyte, is then dissolved in collagen hydrogel.
Wherein, in step b, fibrocellular preparation comprises the steps: fibrocyte to be dissolved in collagen hydrogel.
Wherein, in step c before plantation, the skeletonization section of de-cell heel string recombinant fiber is connected albumen/cadherin rFN/CDH process.
Present invention also offers the organizational project cell-tendon complex prepared by said method.
Beneficial effect of the present invention:
The present invention adopts based on the method for removing cells of histoorgan and cytoskeletal Dual culture strategy, and this method for removing cells not only effectively removes the cell in heel string but also remains the general form of support.And the microstructure of gained support is obvious, aperture and porosity obviously increase, and are beneficial to increment and the autism growth of cell.The present invention breaks up to fiber, cartilage, osseous tissue direction at targeting induced tissue engineering tendon, and then formation " structure of dividing a word with a hyphen at the end of a line " has comparatively high potential and application prospect.
Accompanying drawing explanation
Fig. 1 shows the preparation of de-cell rabbit heel string support
The general form of heel string before and after (a, b) de-cell;
Before and after (c, e, d, f) de-cell process, heel string H & E dyes (hematoxylin-eosin stains) (× 100);
(g, i, h, j) SEM(scanning electron microscope) horizontal stroke, the longitudinal section electron-microscope scanning result (× 1000 times) of heel string before and after de-cell.
Fig. 2 shows genes amplification type adenovirus vector construct schematic diagram and transfection efficiency
(a) sox-9 and runx-2 high expressed adenovirus vector construct schematic diagram;
B () DNA electrophoresis showed pAdtrack-CMV-GFP-sox-9/RunX-2 shuttle plasmid successfully builds;
C () DNA electrophoresis showed pAdEasy-1-sox-9/RunX-2 plasmid successfully builds;
(d, f) pAdEasy-1-sox-9 plasmid is to the high-efficiency transfection of chondrocyte;
(e, g) pAdEasy-1-RunX-2 is to osteoblastic high-efficiency transfection.
The preparation of engineering tendon external structure principle that Fig. 3 shows " structuring of dividing a word with a hyphen at the end of a line ", de-cytoskeletal filament compatibility detection, collagen hydrogel and rFN/CDH
(a) " structuring of dividing a word with a hyphen at the end of a line " engineering tendon external structure schematic diagram;
(, b) " structuring of dividing a word with a hyphen at the end of a line " engineering tendon function division: FB is for becoming zone of fiber, CH for becoming cartilaginous areas, OS skeletonization region;
Collagen hydrogel cell mixture outward appearance before and after (c) gel solidification;
D () mtt assay is capable takes off cytoskeletal cell compatibility and detects, abscissa represent incubation time (my god), vertical coordinate represents that (control and scaffold represents tendon tissue before and after de-cell to optical density (OD) value respectively.）
(e) XPS(X XPS Analysis) confirm that rFN/CDH success is surface-crosslinked with de-cell tendon scaffold;
F () SEM confirms to be adhered at rack surface by the mediated cell success of rFN/CDH.
Fig. 4 shows the expression of gene protein cartogram (* P<0.05, ﹟ P<0.01) of each group of tendon from tissue engineering at the corresponding mark of different times
The mrna expression situation of the skeletonization mark (OCN, OPN and BSP) during (a) RunX2 and 10 day when respectively experiment is grouped in 5 days;
The mrna expression situation of cartilage mark (COL2A1, COMP and Aggrecan) during (b) sox-9 and 10 day when respectively experiment is grouped in 5 days and 10 days;
C () respectively tests the protein expression situation of skeletonization mark (OCN, OPN and BSP) and cartilage (COL2A1, COMP and Aggrecan) mark when being grouped in 2 weeks.
Group I, simple de-cytoskeleton group; Group II, de-cytoskeleton+fibroblast+chondrocyte+osteoblast group; Group III, de-cytoskeleton+fibroblast+SOX-9 genes amplification chondrocyte+osteoblast group; Group IV, de-cytoskeleton+fibroblast+chondrocyte+RUNX-2 genes amplification osteoblast group; Group V, de-cytoskeleton+fibroblast+SOX-9 genes amplification chondrocyte+RUNX-2 genes amplification osteoblast group.
Fig. 5 shows bone/cartilage/fibrous tissue specific staining (cross section, 200 times): (a-e) tendon from tissue engineering OS section Alizarin red staining is analyzed: representative group respectively I-V (specifically with Fig. 4 c); (f-j) tendon from tissue engineering CH section alcian blue dyeing: representative group respectively I-V (specifically with Fig. 4 c)); (k-o) tendon from tissue engineering FB section H & E staining analysis, representative group respectively I-V (specifically with Fig. 4 c)).
Fig. 6 shows bone/cartilage mark immunofluorescence Epidemiological Analysis (cross section, 400 times): (a-e) OS section Bone Gla protein immunofluorescence, representative group respectively I-V (specifically with Fig. 4 c)); CH section II Collagen Type VI immunofluorescence, representative group respectively I-V (specifically with Fig. 4 c)).
Fig. 7 shows tendon from tissue engineering OS section skeletonization mineralising result: (a-e) OS section Mineral nodules distributes, and only have positive discovery in group II, IV, V, arrow is depicted as the calcium phosphorus tendon from tissue engineering of the spherical distribution of OS section surface; (f-j) the relative amount percentage ratio of C, N, O, Ca, P: representative group respectively I-V (specifically with Fig. 4 c), abscissa representative element electron volts in figure, vertical coordinate represents relative amount.
Detailed description of the invention
Organizational project cell-Tendon Tissue Engineering tendon of the present invention, with de-cell heel string for support, the fiber-cartilage-bone physiological structure of simulation anterior cruciate ligament-bone connection site, plantation fibrocyte, osteoblast and chondrocyte are formed.
Building process of the present invention is described in detail below in conjunction with embodiment:
Embodiment 1 takes off the preparation of cell rabbit heel string support
(1) step: asepticly cut rabbit extremity heel string 5 ~ 6cm, adhering tissue removed by machinery, and aseptic PBS cleans repeatedly.Be placed in constant water bath box at profound hypothermia refrigerator freezing 10min (-70 DEG C) and process 10min(37 DEG C), so 5 times repeatedly, be handled as follows successively at 37 DEG C after the abundant rinsing of PBS: 0.5% trypsin vibration 2h, the abundant rinsing 3 times (30min/ time) of PBS removes residual trypsin, 10% triton x-100 vibration 12h, the abundant rinsing 3 times (30min/ time) of PBS removes residual triton x-100, 5% dodecyl sodium sulfate vibration 2h, PBS abundant rinsing 3 times (30min/ time) is to remove residual dodecyl sodium sulfate, finally use 1 × phosphate buffered saline (PBS) process after be de-cell rabbit heel string support needed for this experiment.Observe the character substantially such as the outward appearance of fresh heel string and de-cell heel string, hardness and elasticity respectively, and the capable histology of specimen taken and electron microscopic examination.
(2) result: as shown in Figure 1, heel string quality comparatively hard (a) before de-cell; After de-cell process, heel string aponeurosis (aponeuroses) is complete, matter is soft, good toughness (b); H & E dyes: heel string inner cell nuclear hyperchromatism au bleu before de-cell process, high-visible (c, e, × 100 times); Have no cell component in heel string after de-cell to exist, arrangement of collagen fibers is loosened, and has comparatively concrete dynamic modulus to there are (d, f, × 100 times); SEM: horizontal stroke, the longitudinal section electron-microscope scanning result of heel string before de-cell, visible arrangement of collagen fibers is fine and close, hole less (g, i, × 1000 times); Horizontal stroke, longitudinal section electron-microscope scanning result after de-cell, arrangement of collagen fibers is loosened, and tendon interfascicular tridimensional network obviously (h, j, × 1000 times).
Embodiment 2 takes off the cytoskeletal filament compatibility and detects
Adopt the cell proliferation rate of CCK-8 method assessment fibroblast on de-cell heel string surface and vigor.
(1) step: first de-cell rabbit heel string support 70% soak with ethanol was spent the night with aseptic PBS rinsing after a day.Then support is cut into some small pieces along cross section, be laid in 96 orifice plates, fibroblast strain of next people being originated (3T3, Shanghai Bo Gu) transfers to the rack surface in 96 orifice plates.At 37 DEG C, 5%CO
2cultivate under condition.0,1,2,3,5,7, within 9 days, respectively the CCK-8 of 10% is added to after continuing to hatch 2h in 96 holes, observes color (yellow) change of liquid and detect OD value (n=6) under the BeckmanDU-70 spectrophotometer of 450nm wavelength, and drawing cell proliferation curve.The results are shown in Figure 3.0,1,2,3,4,5,7,9 days, cell amplification and activity analysis, after result display adds CCK-8, the color (yellow) of liquid and OD value increased gradually, illustrate that this de-cytoskeleton is applicable to the propagation of heterogeneous cell and autism grows.
(2) statistical analysis: often organize sample n=6 in experiment, all data mean+SD represent.Difference SPSS10.0 software between each group of data carries out t-distribution check analysis.During P<0.05, there is statistical significance.
(3) result: cell proliferation and the display of activity analysis result, 0,1,2,3,5,7, within 9 days, detect with CCK-8, the color (yellow) of liquid and optical density OD value increase all gradually, tended to be steady at 7 ~ 9 days (Fig. 4), illustrate that this de-cell heel string support and the cell biological compatibility are better, be applicable to cell in de-cell heel string support in growth and amplification.
Embodiment 3 adenovirus vector construct and respectively to the transfection of seed cell
(1) step: get the stable chondrocyte (RAT-CELL-0096 being cultured to 4 ~ 5 generations, fusion rate 80%, Wuhan is primary primary) and osteoblast (RAT-CELL-0055, Wuhan is primary primary), phenol-chloroform method extracts total serum IgE (TaKaRa total RNA extraction reagent box, Dalian), with upstream and downstream primer clone Sox-9(cartilage controlling gene listed by table 1) and RunX-2(skeletonization controlling gene) total length cds.Select PI-Sce I and I-Ceu I restriction enzyme site, the cDNA fragment of Sox-9 and RunX-2 genes of interest is inserted shuttle plasmid pshuttle-CMV plasmid multiple clone site (gene order refers to table 2).Form recombiant plasmid and adopt enzyme action qualification or order-checking qualification.Recombiant plasmid increases, and purification also prepares enough shuttle plasmid pshuttle-CMV containing genes of interest.With PmeI single endonuclease digestion linearisation recombinant shuttle plasmid, electroresis appraisal plasmid is cut open completely.Glue reclaims linearization plasmid, uses in order to cotransformation.Schematic diagram and the detection figure of the carrier built are shown in Fig. 2 a, 2b, 2c.By above-mentioned linearizing shuttle plasmid (about 1 μ g) and viral backbone plasmid (pAdEasy-1, about 100ng, Stratagene, U.S.) be added to turn in competence EP pipe containing E.coli BJ5183 electricity and mix, proceed to electric revolving cup electric shock (1250 ~ 1500V/mm, 5ms).Add the recovery of SOC or LB culture fluid.Get proper volume electroporated after Cell sap be applied in several kalamycin resistance flat boards and cultivate.The bacterium colony (minimum bacterium colony) that next day, picking flat board grew, inoculation LB culture medium (kalamycin resistance) amplification culture.After extracting plasmid by the method for alkaline lysis antibacterial, use gel electrophoresis screening positive clone.Get positive plasmid and be converted into DH5 α Bacillus coli cells amplification antibacterial and plasmid purification.Use PacI enzyme action recombinant virus plasmid, alcohol settling after total Linearization, ddH
2o dissolves.Liposome plasmid (every 4 μ g PacI about need 20 μ L Lipofectamine).Lipofectamine-DNA mixture is added 2 × 10 of stand density about 50 ~ 70%
6transfection
adenoviruspeople's kidney of e1a gene
epithelial cell(293T cell, laboratory is from depositing) is in 25cm
2flask culture bottle, add containing 10% hyclone (FBS after transduction, Hyclone, the U.S.) DMEM complete medium (Hyclone, the U.S.) cellar culture, observation of cell growing state in process, after about 2 weeks, visible cell pathological changes (CPE) occurs, and can be observed green fluorescence (pAdTrack-CMV plasmid contains GFP reporter gene, Stratagene, the U.S.).Tranducin 11 is after 0 ~ 14 day, and collecting cell precipitates, PBS suspendible, multigelation cell, collected after centrifugation supernatant, infects the 293T cell (laboratory is from depositing) of 50 ~ 70% degrees of fusion.Obvious cytopathy is there is after 2 ~ 3 days.Infect latter 3 ~ 5 days, collect virus when 1/3 ~ 1/2 cells float.Collecting cell also prepares viral supernatants.Identify that recombinant adenovirus produces by Western-blot.Appropriate viral supernatants is added and infects the 293T cell that stand density reaches 90%, collect all cells after 3 ~ 4 days, PBS is resuspended, multigelation 4 times, extract and expand row CsCl continuous gradient centrifugal purification after viral supernatants.Continuous 3 dialysis (10mM Tris, pH8.0+2mM MgC+5% sucrose) can remove CsCl substantially, and virus is stored in-80 DEG C.293T cell to be ready in 96 orifice plates (10
4individual/every hole), be the concentration (10 of 8 groups higher by virus liquid dilution by 2%DMEM culture medium
-3~ 10
-10), add the virus liquid 100 μ L after dilution in each hole.Leave and take two rounds in addition and do not add viral dilution liquid, as negative control.Under 37 DEG C of conditions, put into the metamorphosis situation that incubator cultivates observation of cell after 10 days, 96 orifice plates that count occur the number in the hole of CPE, calculate the pathological changes rate of cell.If (the whole pathological changes of the cell under a certain concentration conditions in each hole, then ratio is designated as 1, if acellular pathological changes, then ratio is designated as 0).Computing formula is as follows: T=101 × 10+ (S-0.5) d/mL, (d=Log10 dilution factor, S=each concentrations of cells pathological changes ratio sum).The same method realizes respectively to Secondary Culture 3-5 generation, the chondrocyte of form stable and osteoblastic transfection
(2) result: recombinant shuttle plasmid pAdtrack-CMV-GFP-sox-9/RunX-2 is through KpnIII, HindIII and pAdEasy-1-sox-9/RunX-2 row SDS-PAGE after PacI enzyme action analyzes, result all shows the product consistent with re-set target, size is all at 30.0kbp, and prove to recombinate successfully (Fig. 2 (a-c)).Virus titer is determined as 1 × 10
9pFU/mL.PAdEasy-1-sox-9 and pAdEasy-1-RunX-2 plasmid is transfected in chondrocyte and osteoblast respectively after PacI enzyme action, after cultivating 24h, under inverted fluorescence microscope, observed result display transfection efficiency is respectively 83% and about 87% (Fig. 2 (d-g)).
Table 1 is for the design of primers of Sox-9 and RunX-2 gene clone
The cDNA gene order that this project of table 2 is cloned in order to Sox-9 and RunX-2
The preparation of embodiment 4 collagen hydrogel
First the acid-soluble Corii Bovis seu Bubali skin type i collagen (Sigma of 100mg is dissolved, the U.S.) (prepare with aseptic double-distilled water in the aseptic acetic acid solution of 40mL0.1%, 220 μm of bacterial filter filtration sterilizations) middle Preparation and storage liquid (solution A), then NaOH and the 10 × DMEM(Hyclone of 0.34M is prepared respectively, the U.S.) liquid, after 220 μm of bacterial filter filtration sterilizations, the ratio of 1:2 is by the NaOH of 0.34M and 10 × DMEM liquid mixing (solution B) by volume, the volume ratio of finally solution A and solution B being pressed 4:1 mixes (solution C), after 220 μm of bacterial filter filtration sterilizations, the penicillin/streptomycin adding 1% in super-clean bench in solution C is the collagen hydrogel solution needed for this experiment.This hydrogel is applied to the excipient of seed cell in support, and to avoid influencing each other caused by cell migration, preparation effect is shown in Fig. 3 d.
The preparation of skeletonization/chondrocyte collagen hydrogel suspension:
(1) osteoblast (6 × 10 of cell fusion degree about 90% is got
6individual), observe under inverted microscope: get a clear view, pollution-free;
(2) discard culture in glassware base, add PBS, wash cell gently, discard PBS;
(3) add 1mL0.25% trypsinization and be about 1min;
(4) add the high sugared complete medium of 2mL10%FBS DMEM/F12 and stop digestion, and with suction pipe, cell is blown down, transfer to centrifuge tube, 500rpm, centrifugal 5min;
(5) the white cell precipitation at the bottom of centrifugal rear visible pipe, adds 1mL collagen hydrogel re-suspended cell and obtains unicellular collagen hydrogel suspension;
(6) cell counting is 1 × 10
6individual/mL.
Embodiment 5 " structuring of dividing a word with a hyphen at the end of a line " engineering tendon external structure
(1) step: cell rabbit heel string support (length 40mm) will be taken off and spend the night with aseptic PBS rinsing with after 70% soak with ethanol 48h.One end (length is about 10mm) of every group (n=4) support is all placed in the DEME/F12 culture medium (Hyclone containing rFN/CDH50 μ g/mL, the U.S.) middle incubated at room 4h, (rFN/CDH preparation method refers to national inventing patent to make rFN/CDH fully be adsorbed in rack surface, " fusion rotein of fibronectin and Calcium ionorphore-11; preparation method and application ", the patent No.: ZL200910103100.6).
To FB+sox-9-CC+RunX-2-OB experimental group: first the bracket end of soaking rFN/CDH being placed in density is 1 × 10
6in the high sugared complete medium suspension of RunX-2-OB, 10%FBS, DMEM/F12 of individual/mL, quiescent culture spends the night.Second day, get 1mL2 × 10 respectively
6the fibroblast of individual/mL or 1 × 10
6the sox-9-CC or 1 × 10 of individual/mL
6the RunX-2-OB collagen hydrogel suspension of individual/mL, becomes fiber with the FB(that 1mL syringe is injected into support along longitudinal stent axis) section, CH(become cartilage) section and OS(skeletonization) section.After hydrogel solidifies completely, tendon scaffold is placed in containing 10% hyclone, 1.5mg/mL β-phosphoglycerol, 100u/mL penicillin, l00g/mL streptomycin, the high sugared culture fluid of DMEM/F12 of TGF-P10ng/mL, ascorbic acid 50 μ g/mL, quiescent culture quiescent culture 2 weeks, is the ligament substitute constructed by this experiment.
Simple support group respectively walks with the high sugared complete medium of the 10%FBS DMEM/F12 of experimental group equivalent or collagen hydrogel process; To the group II respectively same experimental group of step; Group III is except osteoblast section only uses the high sugared complete medium of equivalent 10%FBSDMEM/F12 or collagen hydrogel process, and all the other respectively walk same experimental group; Group IV is except chondrocyte section only uses the collagen hydrogel process of equivalent, and all the other respectively walk same experimental group.
(2) result: take off cell technology by standard and successfully build engineering tendon scaffold, the enhancing transformation that one-tenth cartilage and the osteogenesis gene of seed cell are expressed is realized smoothly by genetic engineering means, and then application collagen hydrogel and the short sticky means of rFN/CDH recombiant protein by seed cell layering plantation, dimensional culture in tendon from tissue engineering, successfully simulate the transition tissue structure of normal ligament-bone junction.
The gene expression of cartilage/skeletonization mark in embodiment 6 tendon from tissue engineering
Select Sox-9 to be early stage cartilage mark, RunX-2 is early stage skeletonization mark; COL2A1(collagen2A1, COL2A1), COMP (Cartilage oligomeric interstitial protein, cartilage oligomeric proteose) and Aggrecan(chondroproteoglycan) be cartilage mark in late period; OCN(osteocalcin, Bone Gla protein), OPN(osteopontin, osteopontin), BSP(Bone Sialoprotein, bone sialoprotein) be late osteogenic mark.
(1) total serum IgE is extracted as follows: when one week and two weeks, tissue is added liquid nitrogen respectively and grind, move in 1.5mLEP pipe, add 1mL TRNzol, cracking 20min on ice, add chloroform 1/5 volume (0.2mL), use whirlpool condition to be vibrated at mixing is placed on 4 DEG C, then use 15000 turns/5 minutes centrifugal; Next upper strata aqueous phase (about 400 μ L) is proceeded in the EP pipe of another 1.5mL; After adding the isopropyl alcohol (about 400 μ L) of same volume, vibration mixing; Under 4 DEG C of conditions, put into centrifuge 15000 turns/20 minutes centrifugal; Sop up supernatant with suction pipe, add in advance with 75% ethanol 1mL of ice cube refrigeration; Put into centrifuge 15000 turns/5 minutes centrifugal; Sop up supernatant with suction pipe, add the ethanol 1mL of 100%; Under 4 DEG C of conditions, put into centrifuge 15000 turns/5 minutes centrifugal; Sop up supernatant with suction pipe, place at airport and within 5-10 minute, make it dry; Then put into the DEPC water of 40 μ L1 ‰ ,-70 DEG C save backup.Get 2 μ L RNA and diluted 50 times, ultraviolet spectrophotometer measures ultraviolet 260nm/280nm place OD value ratio, and acquired results is multiplied by 50, is RNA actual concentrations.Get a part of RNA extracted at random and carry out gel electrophoresis, make glue rear electrophoresis.1g agarose is joined in the TAE buffer of 100mL, puts into microwave oven heating and make it dissolve, be then cooled to 60 DEG C after dissolving, add 5 μ L EB dyestuffs, be finally poured into and be plugged in the glue groove of comb, stand-by after its cooled and solidified.Carried RNA is got 1 μ g electrophoresis, 5V/1cm electrophoresis.According to the primer designed by table 2, adopt " relative quantification " method (Relative Quantitation) of ABI7500 system, real-time fluorescence relative quantification detects the expression conditions of the early stage and late-stage markers thing such as cartilage, bone.Reaction terminates rear confirmation amplification, solubility curve, carries out relative quantitative assay: Δ Ct=genes of interest Ct value-GAPDH Ct value by 2-Δ Δ Ct analytic process;-Δ Δ Ct=Normal group Δ Ct meansigma methods-each sample Δ Ct, 2-Δ Δ Ct reflect the relative expression levels of each sample relative Normal group sample genes of interest.
(2) statistical analysis: often organize sample n=4 in experiment, all data mean+SD represent.Difference SPSS10.0 software between each group of data carries out one factor analysis of variance.During P<0.05, there is statistical significance.
(3) interpretation of result:
Adopt with relative quantification real-time fluorescence RT-PCR methods analyst the expression of the extremely mRNA of downstream marker molecule of cartilage/skeletonization controlling gene sox-9 and RunX-2 gene in tendon from tissue engineering-cellular system engineering tendon.Result is as shown in Fig. 4 (a), to organize the expression calibration of I (simple support group) for 0, the RunX-2mRNA expression then organizing V is 2.91 times (P<0.05) of group II and 6.77 times (P<0.01) of group IV, has significant significant difference.Result is as shown in Fig. 4 (b), and the recruitment that the sox-9mRNA of group V expresses is group II and 195% and the 743%(P<0.05 that organize III respectively), there is significant statistical significance (P<0.05).
Corresponding with it, the classical Downstream regulatory molecule (COL2A1 of sox-9 when the present invention have detected cultivation 10 days, COMP and Aggrecan) and the classical Downstream regulatory molecule (OCN of RunX-2, OPN and BSP), result is as Fig. 4 (a-b) display, cartilage mark COL2A1 is 2.55 times of group II in the expression of group V, organizes IV 9.44 times, all has significant statistical significance (P<0.05); 1.15 times of group III, not statistically significant (P=0.13); The mrna expression rule of COMP with Aggrecan is similar to the mrna expression rule of COL2A1.
To the assessment result display that osteogenesis gene is expressed, when comparing with c group with b group, OCN is 139% and 409%(P<0.05 of group II and group III at the increment of the mrna expression of group V respectively); The mrna expression not statistically signigicant of OCN between group V and group IV; The mrna expression rule of OPN with BSP is similar to OCN.
Above data illustrate, in cultivation framework's engineering tendon of two weeks, the mrna expression of the extremely classical Downstream regulatory molecule of sox-9 and RunX-2 all obviously increases, and confirms that the skeletonization-one-tenth cartilage ability of tendon from tissue engineering is significantly improved thus.
Table 2 is in order to the design of primers of cartilage, the gene expression of bone marker molecule
The protein expression of cartilage/skeletonization mark in embodiment 7 tendon from tissue engineering
1, adopt the method in embodiment 7 to be shredded by the cultivation complex tissue ligament of two weeks, thoroughly homogenate, filtration.
2, total protein is extracted by the following method:
3 μ L aprotiniies are added, 10 μ L0.1M PMSF(Phenylmethanesulfonyl fluorides by 1mL lysate) and 5 μ L0.1MNa
3vO
4(sodium vanadate) (PMSF is shaken up when not having crystallization and just can add lysate) for subsequent use.Add in bottle in the ratio of 100-200 μ L lysate/mL by the liquid prepared, be put in and make its cracking 30min on ice, bottle often will be blown and beaten or is shaken with abundant cell lysis.After cracking is complete, wash piping and druming liquid gently with dropper, then with dropper, lysate is moved to (whole operation is carried out on ice) in 10mL centrifuge tube as far as possible.The centrifugal 15min of 12000rpm (opening centrifuge pre-cooling in advance) at 4 DEG C.Centrifugal complete after, get supernatant and load in EP pipe, get a part and survey concentration ,-80 DEG C of preservations, appropriate subpackage.Not repeatedly by its freeze thawing.
3, the content of albumen is measured:
(1) making of standard curve;
(2) BSA(1mg/mL is taken out from-20 DEG C), after room temperature is melted, for subsequent use;
(3) by table 3 deionized water, BSA is diluted to following concentration (mg/mL):
The dilution ratio of table 3BSA
| BSA(mL) | 0 | 1 | 2 | 4 | 6 | 8 | 10 |
| Deionized water (mL) | 10 | 9 | 8 | 6 | 4 | 2 | 0 |
(4) often pipe adds 100 μ L determination of protein concentration working solutions (micro-BCA, Pierce, the U.S.)
(5) in 37 DEG C of water-baths, 30min is placed.
(6) UV detector in 562nm wavelength measures absorbance
(7) computer produces standard curve automatically, and standard curve is preserved backed off after random.
4, the detection of the protein content of sample
(1) by a certain percentage by Sample Dilution;
(2) BCA reagent B:A=1:50;
(3) diluted sample and BCA working solution ratio are 1:10;
Place 30 minutes in the water bath of (4) 37 DEG C;
(5) UV detector in 562nm wavelength measures absorbance;
(6) experimental data is preserved.
5, SDS-PAGE electrophoresis
Make separation gel:
(1) destination protein molecular size range is: COMP(105KD), select the separation gel of 8%.BSP(35KD), the separation gel of 12% is selected.OPN(55KD), the separation gel of 10% is selected.Aggrecan (200KD), selects the separation gel of 6%.COL2A1(190KD), the separation gel of 6% is selected.OCN (35KD), selects the separation gel of 12%.GAPDH (37KD), selects the separation gel of 12%;
(2) concentration of concentrated glue is made as 5%;
(3) cleaning glass plate, put into clamping plate after alignment glass plate and clamp, in order to prepare encapsulating, it vertically being blocked on the top of the shelf;
(4) after fully mixing in above ratio, each for separation gel component is poured in glass plate holder seam;
(5) after crack being closed wait 20 ~ 30min with n-butyl alcohol.N-butyl alcohol is outwelled, is rinsed twice with deionized water;
(6) manufacture concentrated glue with reference to the ratio of 5%, add after mixing and insert comb immediately;
(7) wait for that 30min ~ 1h takes out comb, assembling electrophoretic apparatus.In electrophoretic apparatus, add electrophoresis liquid, first add half inclined to remove bottom bubble;
(8) sample treatment: get appropriate protein sample (according to detection sample protein content), add appropriate 5 × SDS Loading Buffer, boil 3-5min in boiling water;
(9) loading after room temperature is cooled to.Generally stay first hole as maker, vacant hole adds appropriate 1 × SDS Loading Buffer pressure zone;
(10) electrophoresis: 1., 30V runs five minutes, 3., 120V separation gel (can make 120V into after there is marker) 2., 80V concentrates glue;
(11) isolated edge is gone to when being about 0.5cm, powered-down;
(12) transferring film: the electricity that first falls turns liquid in the flat pallet of 2.5L, foam-rubber cushion and filter paper is dipped in electricity and turns in liquid.The film that our laboratory uses is PVDF(polyvinylidene fluoride) film, cut one jiao of film after taking out by custom and labelling, then put into absolute methanol immersion 5-10s.Then take out and be put in electricity and turn immersion 5min in liquid, the size according to object band cuts adhesive tape.Lie against on pvdf membrane, after alignment, filter paper is spread.Then clip clip, note the correct order (both positive and negative polarity) of assembling.100v, 1.5 ~ 2h, can add ice chest when transferring film, and use magnetic stirrer can be used to ensure that film system is at lower state of temperature, prevents transferring film effect to be affected.
6, immunoblotting
(1) Western-blot method semi-quantitative analysis COL2A1, COMP, Aggrecan, OCN, BSP, OPN protein expression situation (n=3).Use goat-anti COMP, Aggrecan (1:1000dilution, Sigma) antibody; Anti-BSP, OCN (1:1000dilution, the Sigma) antibody of rabbit; And GAPDH, OPN, COL2A1 (1:1000dilution, Sigma) antibody is primary antibodie, the anti-goat-anti body (1:1000dilution, Sigma) of two anti-use rabbits; Goat anti-rabbit antibodies (1:1000dilution, Sigma); And sheep anti-mouse antibody (1:1000dilution, Sigma) be two resist.
(2) image analysis software is used to measure the gray scale ratio of band, parallel statistical analysis.Often organize sample n=4 in experiment, all data mean+SD represent.Difference SPSS10.0 software between each group of data carries out one factor analysis of variance.During P<0.05, there is statistical significance.
(3) interpretation of result:
The protein translation level of COL2A1, COMP, Aggrecan, OCN, OPN and BSP in tendon from tissue engineering protein extract is have detected by immunoblotting.Result is as shown in Fig. 4 (c), and cartilage mark COL2A1 is 1.87 times of group II at the protein expression of group V, and organize IV 2.8 times, both all have significant statistical significance (P<0.05); Not statistically significant (1.12 times, P>0.05) between group V and group III, the expression of COMP with Aggrecan is similar to COL2A1.
Skeletonization mark OCN the protein expression of group V comparatively organize II and group III add respectively 230% and 407%, there is significant statistical significance (P<0.05); Not statistically significant (P>0.05) between group V and group IV, the protein expression rule of OPN with BSP is similar to OCN protein expression rule.
Cartilage/skeletonization in embodiment 8 tendon from tissue engineering/one-tenth fibrous tissue qualification
1, particular tissues dyeing
(1) after the cultivation composite tissue engineering ligament of 2 weeks being given PBS rinsing, 10% paraformaldehyde is fixed, paraffin embedding, 5um thickness serial section (cross section) row immunofluorescence and histological stain (n=4) afterwards.According to reagent description, observe the continuous distribution situation of fibroblast and fibrous tissue, GAGs and calcium tuberosity in composite tissue engineering ligament respectively with Hematoxylin-eosin, alcian blue and Alizarin red staining.
(2) Image Acquisition: the section made is placed in common light microscopic basis of microscopic observation and obtains original image.
(3) interpretation of result:
Alizarin red staining result shows, the skeletonization Duan Jun of group V and group II, IV is dyed to salmon pink (Fig. 5 (a-e)), but, obvious salmon pink visible (result does not provide) is not had in CH and FB region and other each group, the generation primary limitation of calcium tuberosity is in OS region, and supposition is the secretion that the osteoblast increased accelerates representative extracellular matrix.But compared with group II, group V and group IV have the generation of more mineral tendon from tissue engineering, and this result shows RunX-2 gene transfection and accelerates corresponding osteoblastic one-tenth bone mineralization.
Alcian blue coloration result finds, positive staining (blueness) mainly concentrate on group V, group II and group III and CH region (Fig. 5 (f-j)), the dyeing of group V and group IV is obviously better than group II, and this result describes the GAGs generation that sox-9 facilitates chondrocyte.
H & E coloration result shows the distribution (Fig. 5 (k-o)) in FB section of fibroblast and collagen fiber, compared with group I, cell and the newly-generated extracellular matrix of amplification cover the surface being interconnected hole, have no obvious Morphological Differences between group II-V.
2, immunofluorescence analysis
(1) after the cultivation composite tissue engineering ligament of 2 weeks being given PBS rinsing, 10% paraformaldehyde is fixed, paraffin embedding, 5um thickness serial section (cross section) row immunofluorescence and histological stain (n=4) afterwards.According to reagent description, detect Bone Gla protein in tendon from tissue engineering, the expression and distribution situation of COL2A1, with mouse anti human Bone Gla protein (osteocalcin, OCN, diluted concentration is 1:100, and the anti-human COL2A1(collagen2A1 of rabbit abcam), COL2A1, diluted concentration is 1:50, abcam) cut into slices as primary antibodie tagged tissue, with the donkey anti-mouse of cy3 labelling, (diluted concentration is for 1:50, abcam) and FITC(Fluorescein isothiocyanate) (diluted concentration is 1:100 for the donkey anti-rabbit of labelling, abcam) fluorescent antibody be two resist, nucleus DAPI(4', 6-diamidino-2-phenylindone) dyeing (blueness).
(2) Image Acquisition: the section made is placed in fluorescence microscopy Microscopic observation and obtains original image.
(3) the red painted areas of interpretation of result: Fig. 6 (a-e) beam is OCN fluorescence mark region, and blueness is nucleus, and the OCN expression of semi-quantitative results prompting group V, apparently higher than all the other each group, illustrates that tendon from tissue engineering is the strongest at the osteogenic ability of each group.Fig. 6 (f-j) viride nitens painted areas is OCN fluorescence mark region, and blueness is nucleus, and the COL2A1 expression of semi-quantitative results prompting group V, apparently higher than all the other each group, illustrates that tendon from tissue engineering is the strongest the one-tenth cartilage ability of each group.
3, SEM and EDX of tendon from tissue engineering OS section surface analyzes
(1) after the Dual culture composite tissue engineering ligament of 2 weeks being given PBS rinsing three times, 24 ~ 48 hours are fixed with 2.5% glutaraldehyde, graded ethanol: dehydration 10 minutes and tertiary propanol in the 10 minutes → dehydrated alcohol that dewaters in dehydration 10 minutes → dehydrated alcohol in dehydration 10 minutes → 90% ethanol in dehydration 10 minutes → 70% ethanol in 10 minutes → 50% ethanol that dewaters in 30% ethanol: dehydration dehydration rear (noting changing liquid process in this process should not stop too of a specified duration at every turn in atmosphere) in 10 minutes in dehydration 10 minutes → anhydrous tertiary butanol in dehydration 10 minutes → anhydrous tertiary butanol in 10 minutes → 90% tert-butyl alcohol that dewaters in dehydration 10 minutes → 70% tert-butyl alcohol in 10 minutes → 50% tert-butyl alcohol that dewaters in 30% tert-butyl alcohol, specimen is fixed in Electronic Speculum holder and utilizes energy dissipation x-ray spectroscopy instrument (EDX, Horiba EX-220) sxemiquantitative tendon from tissue engineering surface calcium, after the distribution situation of the mineralising elements such as phosphorus, take out sample carrier and to the mode of appearance being placed in tissues observed engineering tendon under scanning electron microscope (S-3400N) after rack surface gold-plated (Hitachi E-1030ion sputterer) again.
(2) statistical analysis: often organize sample n=4 in experiment, all data mean+SD represent.Difference SPSS10.0 software between each group of data carries out t-distribution check analysis.During P<0.05, there is statistical significance.
(3) interpretation of result:
After In vitro culture 2 weeks tendon from tissue engineering, effects on surface element detects, result is as shown in Figure 7: in the secure execution mode (sem except group I and group III (Fig. 7 (a-e)), what all the other respectively organized the surperficial all visible extensively distribution of tendon from tissue engineering is the mineralized material that nodositas or patch shape distribute, especially the highest to organize V performance, these materials are limited in and have adhered to osteoblastic OB section surface by rFN/CDH.
In addition, by EDX, the elementary composition of each group of sample surface is analyzed, OS section surface detects the accurate distribution (Fig. 7 (f-j)) of calcium and phosphorus, find that calcium phosphorus content is all significantly higher than group II and the group IV of non-row RunX-2 gene delivery in group V and group III, the efficient RunX-2 transfection also demonstrated thus in tendon from tissue engineering accelerates the Osteoblast Differentiation effect of respective regions simultaneously.Each group of calcium phosphorus relative weight percents is as shown in labelling in figure.
Claims (13)
1. the vitro construction method of bionics ligament-bone connector, is characterized in that: comprise the steps:
The preparation of a, de-cell heel string;
The preparation of b, seed cell: comprise fibrocyte, the chondrocyte of Sox-9 genes amplification, the osteoblastic preparation of Runx-2 genes amplification and gene transfection;
C, plantation: de-cell heel string is divided into into segment of fiber FB-according to its longitudinal axis successively with transverse axis trend and becomes cartilage section CH-skeletonization section OS tri-26S Proteasome Structure and Function regions, the chondrocyte layering plantation of the osteoblast of fibrocyte, Runx-2 genes amplification, Sox-9 genes amplification, segmentation are planted into segment of fiber, become cartilage section, skeletonization section, after cultivating histology's checking in 5 ~ 7 days, then cultivate use in 1 week; Described layering plantation refers to is transplanted to center, transverse section by heel string surface, makes the osteoblast of fibrocyte, Runx-2 genes amplification, interior, domestic and abroad each 1/3 region that the chondrocyte of Sox-9 genes amplification lays respectively at transverse section.
2. the method for claim 1, is characterized in that: in step a, de-cell heel string support is the heel string tissue deriving from rabbit, dog, cattle.
3. method as claimed in claim 1 or 2, is characterized in that: in step b, osteoblastic preparation comprises the steps: the adenovirus vector of construction expression early stage skeletonization mark RUNX-2, transfection osteoblast.
4. method as claimed in claim 1 or 2, is characterized in that: in step b, the preparation of chondrocyte comprises the steps: the adenovirus vector of construction expression early stage cartilage mark SOX-9, transfection chondrocyte, is then dissolved in collagen hydrogel.
5. method as claimed in claim 3, is characterized in that: in step b, the preparation of chondrocyte comprises the steps: the adenovirus vector of construction expression early stage cartilage mark SOX-9, transfection chondrocyte, is then dissolved in collagen hydrogel.
6. method as claimed in claim 1 or 2, is characterized in that: in step b, fibrocellular preparation comprises the steps: fibrocyte to be dissolved in collagen hydrogel.
7. method as claimed in claim 3, is characterized in that: in step b, fibrocellular preparation comprises the steps: fibrocyte to be dissolved in collagen hydrogel.
8. method as claimed in claim 4, is characterized in that: in step b, fibrocellular preparation comprises the steps: fibrocyte to be dissolved in collagen hydrogel.
9. method as claimed in claim 1 or 2, is characterized in that: in step c before plantation, the skeletonization section of de-cell heel string recombinant fiber is connected albumen/cadherin rFN/CDH process.
10. method as claimed in claim 3, is characterized in that: in step c before plantation, the skeletonization section of de-cell heel string recombinant fiber is connected albumen/cadherin rFN/CDH process.
11. methods as claimed in claim 4, is characterized in that: in step c before plantation, the skeletonization section of de-cell heel string recombinant fiber is connected albumen/cadherin rFN/CDH process.
12. methods as claimed in claim 6, is characterized in that: in step c before plantation, the skeletonization section of de-cell heel string recombinant fiber is connected albumen/cadherin rFN/CDH process.
The 13. organizational project cell-tendon complex prepared by method described in any one of claim 1 ~ 12.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310462274.8A CN103638558B (en) | 2013-09-30 | 2013-09-30 | In vitro construction method for bionic ligament-bone tissue engineering connector |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310462274.8A CN103638558B (en) | 2013-09-30 | 2013-09-30 | In vitro construction method for bionic ligament-bone tissue engineering connector |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103638558A CN103638558A (en) | 2014-03-19 |
| CN103638558B true CN103638558B (en) | 2015-04-29 |
Family
ID=50243927
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310462274.8A Active CN103638558B (en) | 2013-09-30 | 2013-09-30 | In vitro construction method for bionic ligament-bone tissue engineering connector |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103638558B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111449804B (en) * | 2020-03-18 | 2022-12-20 | 中南大学湘雅医院 | Tissue engineering scaffold mineralization method |
| CN114732957B (en) * | 2022-03-16 | 2022-12-23 | 武汉理工大学 | Mineralized tendon composite material with prestress and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101748095A (en) * | 2008-11-28 | 2010-06-23 | 上海中医药大学附属普陀医院 | Method for directionally inducing cartilage cells |
| CN102614546A (en) * | 2011-01-26 | 2012-08-01 | 香港中文大学 | Cell sheet for tissue repair and bio-artificial tissue engineering, method of producing the same and method of using the same |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2484751B1 (en) * | 2002-04-16 | 2018-11-28 | Princeton University | Method of analysing polynucleotides |
| US20110252486A1 (en) * | 2008-12-18 | 2011-10-13 | Osaka University | Chondrocyte-like cell, and method for producing same |
-
2013
- 2013-09-30 CN CN201310462274.8A patent/CN103638558B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101748095A (en) * | 2008-11-28 | 2010-06-23 | 上海中医药大学附属普陀医院 | Method for directionally inducing cartilage cells |
| CN102614546A (en) * | 2011-01-26 | 2012-08-01 | 香港中文大学 | Cell sheet for tissue repair and bio-artificial tissue engineering, method of producing the same and method of using the same |
Non-Patent Citations (4)
| Title |
|---|
| In vitro generation of osteochondral differentiation of human marrow mesenchymal stem cells in novel collagen-hydroxyapatite layered scaffolds;Jiaan Zhou等;《Acta Biomaterialia》;20110630;第7卷;3999-4006 * |
| Involvement of SOX-9 and FGF-23 in RUNX-2 regulation in osteoarthritic chondrocytes;Timoklia Orfanidou等;《J. Cell. Mol. Med.》;20091231;第13卷(第9B期);3186-3194 * |
| 半月板组织工程研究进展;符培亮等;《中国修复重建外科杂志》;20130430;第27卷(第4期);486-491 * |
| 脱细胞跟腱复合人成纤维细胞共培养的实验研究;王直兵等;《中国修复重建外科杂志》;20130731;第27卷(第7期);805-809 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103638558A (en) | 2014-03-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yin et al. | Induction of mesenchymal stem cell chondrogenic differentiation and functional cartilage microtissue formation for in vivo cartilage regeneration by cartilage extracellular matrix-derived particles | |
| Izuta et al. | Meniscal repair using bone marrow-derived mesenchymal stem cells: experimental study using green fluorescent protein transgenic rats | |
| CN104353111B (en) | A kind of bioprosthetic material for abdominal-wall defect and preparation method thereof | |
| CN105963785B (en) | Acellular matrix material based on adipose-derived stem cell membrane and preparation method thereof | |
| CN102174468A (en) | Method and application for inducing human umbilical cord mesenchyme stem cells to be differentiated into testicular interstitial cells | |
| Li et al. | Tissue-engineered mesh for pelvic floor reconstruction fabricated from silk fibroin scaffold with adipose-derived mesenchymal stem cells | |
| Beitzel et al. | The future role of mesenchymal stem cells in the management of shoulder disorders | |
| CN101263224A (en) | Use of adipose tissue-derived stromal stem cells in treating fistula | |
| CN110237303A (en) | The preparation method of the Acellular bone membrane matrix gel rubber material in natural tissues source | |
| CN104974984A (en) | Adipose tissue-derived mesenchymal stem cell amplification culture method | |
| Farag et al. | Assessment of static and perfusion methods for decellularization of PCL membrane-supported periodontal ligament cell sheet constructs | |
| CN110478528B (en) | Preparation method and application of novel tissue repair promoting material | |
| CN111603610B (en) | Preparation method of adipose tissue extracellular matrix | |
| CN102861360B (en) | Neural restoration promoting material as well as preparation method and application thereof | |
| Alves da Silva et al. | Electrospun nanofibrous meshes cultured with Wharton's jelly stem cell: an alternative for cartilage regeneration, without the need of growth factors | |
| Li et al. | Bone marrow enriched graft, modified by self-assembly peptide, repairs critically-sized femur defects in goats | |
| CN109847098A (en) | A kind of compound bio timbering material for repairing bone defect | |
| CN107376025B (en) | Preparation method and application of cell-scaffold composite material for cartilage injury repair | |
| CN108245708A (en) | A kind of induction regenerated bioactive bracket of tendon tissue and its preparation method and application | |
| Li et al. | Construction of engineering adipose‐like tissue in vivo utilizing human insulin gene‐modified umbilical cord mesenchymal stromal cells with silk fibroin 3D scaffolds | |
| Zheng et al. | Ovary-derived decellularized extracellular matrix-based bioink for fabricating 3D primary ovarian cells-laden structures for mouse ovarian failure correction | |
| CN103638558B (en) | In vitro construction method for bionic ligament-bone tissue engineering connector | |
| CN106474156A (en) | Purposes in preparing medicine for the compositionss | |
| Zhou et al. | Meniscus regeneration with multipotent stromal cell therapies | |
| CN107119020A (en) | A kind of hepatic injury targeting mescenchymal stem cell based on miR 9 and preparation method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |