CN110507852A - A kind of osseous tissue renovating material - Google Patents
A kind of osseous tissue renovating material Download PDFInfo
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Abstract
The invention belongs to organizational project Regeneration and Repair field, disclosing a kind of osseous tissue renovating material includes nano-particle modified more micropore cytoskeletons and cell factor.Nano-particle modified more micropore cytoskeletons in osseous tissue renovating material of the present invention can high-efficient carrier cell factor SDF-1 and BMP-2, and there is certain slow release effect to the cell factor of load.Nano-particle modified more micropore cytoskeletons have good biocompatibility and cell adhesive ability.Nano-particle modified more micropore cytoskeletons are by loading and discharge SDF-1, obtaining superior chemotactic and raising the activity of self seed cell；By loading BMP-2, changes local microenvironment, induce MSCs Osteoblast Differentiation.The synergistic effect for two cell factors that osseous tissue renovating material of the present invention is loaded by nano-particle modified more micropore cytoskeletons, can solve the demand of seed cell and extracellular microenvironment simultaneously.
Description
Technical field
The invention belongs to organizational project Regeneration and Repair fields, and in particular to a kind of osseous tissue renovating material.
Background technique
Congenital abnormality, severe trauma, osteogenesis imperfecta and malignant tumour expand the illness such as operation excision and are likely to result in
Extensive tis-sue defect, and Repair of tissue defect reconstruction is a great problem that current reconstructive surgery doctor is faced, it is close with it
Relevant regeneration and restoration research be also Current Basic Medicine research field most broad development prospect research direction it
One.And since bone tissue in anatomical structure and function mainly supports and protect its hetero-organization internal organs, such as wall of the chest bone, hold
The microcosmic physiological function carried is relatively fewer, and bone tissue reparation reconstruction only needs to restore its dissection support construction and is able to satisfy substantially
Functional requirement.It can be seen that the reconstruction of bone defect repair is in current regeneration and restoration research closest to clinical application
Direction.
In clinical practice, in face of the bone defect of larger size, surgeon needs timely and effective in rebuilding prosthesis
Ground is replaced and is implanted into the bony structure of defect, its structure and physiological function are restored to the greatest extent.Traditional surgery
Bone Reconstruction Strategy mainly includes the use of autologous bone transplanting, allograft and artificial implantation.Autograft makees
For the goldstandard of bone graft, there is optimal tissue compatible sexual clorminance, but there is materials are limited, and surgical procedure is more complex etc.
Disadvantage is not suitable for large-scale bone defect reconstruction.Compared with autotransplantation, allograft transplanting exists immune
The relevant risk of reaction and transmission of infection, and artificial implantation needs more for the individual in growth and development stage
Secondary reconstruction operations are to adapt to the demand of body growth.With the quick hair of regenerative medicine and Tissue Engineering Study field in recent years
Exhibition, the reconstruction of organizational engineering Bone Defect Repari are considered as most potential alternative solution, and organizational engineering Bone Defect Repari, which is rebuild, has it
Unique advantage, such as can infinitely supply, no rejection, avoid transmission and can self-adaptive growth development demand
Deng.The key element of bone tissue engineer is the seed cell of Regeneration and Repair and for cell adherence, proliferation and the bracket of differentiation material
Material.Previous research strategy often fails to take into account seed cell, bracket and the more a elements of ECM.
Summary of the invention
In view of this, providing a kind of bone tissue reparation it is an object of the invention to aiming at the problems existing in the prior art
Material, to solve the demand of seed cell and extracellular microenvironment.
To achieve the purpose of the present invention, the present invention adopts the following technical scheme:
A kind of osseous tissue renovating material, which is characterized in that including nano-particle modified more micropore cytoskeletons and cell
The factor.
Nano-particle modified more micropore cytoskeletons are by positively charged described in osseous tissue renovating material of the present invention
The lotus and nano particle that can be chemically crosslinked is combined into the more micropore cell scaffold materials of gelatine-chitosan-agarose.
Preferably, nano particle described in nano-particle modified more micropore cytoskeletons is chitosan oligosaccharide/heparin
Nano particle.
Preferably, gelatine-chitosan-agarose described in nano-particle modified more micropore cytoskeletons is more
Micropore cell scaffold material is prepared by chitosan, gelatin and agarose using chemical crosslinking freezing gel method, is stated clearly
Glue-chitosan-agar sugared content is 4~5wt%, and the mass ratio of the chitosan, gelatin and agarose is 1:1:(2~3).
In some embodiments, the chemical crosslinking freezing gel method prepares the more micropores of gelatine-chitosan-agarose
Cytoskeleton is specially that chitosan is dissolved completely in after acetum and gelatin is added mixes well dissolution, and addition agarose is water-soluble
Liquid mixes, and 5% (v/v) glutaraldehyde solution is added dropwise in blending process and is crosslinked, and is subsequently placed at -14 DEG C and is incubated for
Freezing gel is prepared within 16 hours, thaw at RT prepares more micropore cytoskeletons.
In some embodiments, based on mg/ml, the quality volume of the gelatin and described 5% (v/v) glutaraldehyde solution
Than for 150:1 to 250:1.
In some embodiments, in the osseous tissue renovating material, nano-particle modified more micropore cells
The preparation method of bracket is to obtain nano granule suspension after nano particle is resuspended in water, and gelatin-chitosan-agarose is mostly micro-
Hole cell scaffold material, which is added in nano granule suspension, shakes 6 hours for 37 DEG C, and 5% 37 DEG C of glutaraldehyde reactions are added
20min is freeze-dried after cleaning.
Preferably, cell factor described in osseous tissue renovating material of the present invention is SDF-1 and/or BMP-2.
It as shown from the above technical solution, include nano-particle modified the present invention provides a kind of osseous tissue renovating material
More micropore cytoskeletons and cell factor.Nano-particle modified more micropores in osseous tissue renovating material of the present invention are thin
Born of the same parents' bracket can high-efficient carrier cell factor SDF-1 and BMP-2, and nano-particle modified more micropore cell scaffold materials are to negative
The cell factor of load has certain slow release effect.CSO/H nano-particle modified more micropore cytoskeletons have good life
Object compatibility and cell adhesive ability.Nano-particle modified more micropore cytoskeletons are obtained by loading and discharging SDF-1
Superior chemotactic and the activity for raising self seed cell；Nano-particle modified more micropore cytoskeletons pass through load BMP-
2, change local microenvironment, induces MSCs Osteoblast Differentiation.Osseous tissue renovating material of the present invention passes through nano-particle modified
The load of more micropore cytoskeletons two cell factors synergistic effect, seed cell and extracellular micro- can be solved simultaneously
The demand of environment.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, below will to embodiment or
Attached drawing needed to be used in the description of the prior art is briefly described.
Fig. 1 show more than the 1 micropore cytoskeleton of embodiment macroscopic appearance (A) and SEM image (B, 400 ×, bar=200 μ
m)；More micropore cytoskeletons section shows the irregular class cellular structures of thin-walled under scanning electron microscope；
Fig. 2 shows embodiment 1 by the SEM image of CSO/H nano-particle modified more micropore cytoskeletons；A:10000 ×,
Bar=5 μm；B:25000 ×, bar=3 μm, nano particle is in microspheroidal, is uniformly distributed in more micropore cytoskeletons surface；
Fig. 3 shows embodiment 2WST-8 chemical structure and reaction principle figure；
Fig. 4 shows embodiment 2CCK-8 measurement for assessing the more micropore cytoskeletons or unmodified bracket pair of NP modification
The mode image of the cytotoxic effect of MSC；
Fig. 5 shows 2 group of cells growth curve figure of embodiment, n=5；
Fig. 6 shows the cell attachment evaluation figure of the nano-particle modified more micropore cytoskeletons of embodiment 3；
Fig. 7 shows that embodiment 4 is detected using enzyme-linked immunosorbent assay (ELISA) from nano-particle modified more micropores
The release profiles of the SDF-1 and BMP-2 of cytoskeleton, SDF-1 early stage are in discharge gradually to tend to be steady after outburst release the 4th day,
BMP-2 releasing trend is slow compared to more flattening out for SDF-1, but involves burst size more than SDF-1 in the 10th day beginning BMP-2
(n=3)；
Fig. 8 shows the nano-particle modified more micropore cell branch of embodiment 5Transwell Cell migration assay model evaluation
The mode image of the chemotactic activity SDF-1 of frame release external to MSCs；
Fig. 9 shows that embodiment 5 uses external Transwell cell migration model evaluation from nano-particle modified more micropores
Cytoskeleton discharges the ability of the fixed point migration of SDF-1 induction stem cell, wherein figure (A) is only for SDF-1, cytoskeleton
The presentation graphics of the MSC of transfer have the branch of-NP control and the NP modification of the SDF-1 of load in Transwell measurement
Frame；The average for scheming the translation MSC after (B) is incubated for 12,24,48 and 72 hours in Transwell migration measurement, the result is that coming
From average value ± SD, * p < 0.05, * the * p < 0.01, n=15 in five of the three independent experiments different visuals field (the result is that coming from
The cell number mean value ± SD in the different visuals field of five of three samples)；
Figure 10 shows that embodiment 6MSCs osteogenic induction experiment assesses nano-particle modified more micropore cytoskeletons release
The osteoinductive activity mode image of BMP-2；
Figure 11 shows the active analysis of alkaline phosphatase (ALP) and the madder of MSCs of the embodiment 6 after being incubated for up to 21 days
The result figure of plain red colouring；Figure (A) shows the ALP activity for being inoculated into 24 orifice plates and cultivating together with material up to 21 days MSC
As a result analysis chart is expressed as average value ± SD, every group of p < 0.05 *；Figure (B) shows is inoculated with the MSC's for being up to 21 days on bracket
Alizarin red staining result figure: nanometer-bracket group is incubated with minimal medium, and by positive control (+) group and self-bone grafting
Culture medium is incubated with；
Figure 12 shows the effect of the internal recruitment MSC of the nano-particle modified more micropore cytoskeletons release SDF-1 of embodiment 7
Fruit figure, n=3.
Specific embodiment
The invention discloses a kind of osseous tissue renovating materials.Those skilled in the art can use for reference present disclosure, suitably change
Into realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are aobvious for a person skilled in the art
And be clear to, they are considered as being included in the present invention.Method and product of the invention has passed through preferred embodiment and has carried out
Description, related personnel obviously can not depart from the content of present invention, method described herein is modified in spirit and scope or
Appropriate changes and combinations carry out implementation and application the technology of the present invention.
In some embodiments, bio-compatible of the present invention to nano-particle modified more micropore cell scaffold materials
Property assessment mainly by two aspect progress: first: being quantitatively evaluated using CCK-8 kit measurement nano-particle modified
More micropore cytoskeletons or the not influence by nano-particle modified more micropore cytoskeletons to the cell Proliferation of MSCs；Second:
After being inoculated in nano-particle modified more micropore cell scaffold materials by the MSCs for marking GFP, by being inverted fluorescence microscopy
The presence or absence of the cell of sem observation inoculation attachment, cellular morphology and cell density are nano-particle modified to carry out qualitative evaluation
A possibility that more micropore cell scaffold materials are as cytoskeleton.The results show that nano-particle modified more micropore cell branch
Frame is proliferated without obvious inhibiting effect MSCs, and can effectively adhere to seed cell, has good cell compatibility.GFP mark
The MSCs of note can be attached in nano-particle modified more micropore cell scaffold materials surface and micropore, cell expansion it is star-shaped,
Shuttle shape or mostly prominent shape, cell is in healthy form, and density is higher.Showing nano-particle modified more micropore cytoskeletons is
The good steel framework of MSCs has excellent cell compatibility.
In some embodiments, the present invention is assessed nano-particle modified more using Transwell Cell migration assay
The ability of the SDF-1 of micropore cytoskeleton more micropore cytoskeletons release inducing chemotactic MSCs in vitro.It is clashed by cotton swab poly-
On the upside of carbonic ester film after cell, and with the cell dyeing on the downside of crystallization purple membrane come the migration quantity of quantitative assessment MSCs.As a result it shows
Show after being carried on nano-particle modified more micropore cytoskeletons, SDF-1 can keep its bioactivity for a long time, lasting to send out
Wave the effect of chemotactic stem cell.
In some embodiments, the present invention assesses nano-particle modified more micropores using MSCs osteogenic induction experiment
The BMP-2 of cytoskeleton release induces the ability of MSCs Osteoblast Differentiation in vitro.It is detected by osteogenic induction measuring method from NP and is repaired
The activity of the BMP-2 of the bracket release of decorations.The BMP-2 of nano-particle modified more micropore cytoskeletons load is held as the result is shown
Continuous release may continuously play osteoinductive activity.By alizarin red to the further qualitative evaluation doped calcium of cell dyeing and calcium
The degree of tubercle.The result shows that bracket or nano particle are no apparent active to MSCs Osteoblast Differentiation is promoted.And load cells
Nano-particle modified more micropore cytoskeletons of the factor also show that more significant positive staining as a result, most deep madder is presented
Plain red colouring is as a result, show that nano-particle modified more micropore cytoskeletons and cell factor play significantly in self-bone grafting
Synergy.
In some embodiments, the present invention should imaging system (Spectrum In Vivo using small animal living body
Imaging System, IVIS) evaluation nano-particle modified more micropore cytoskeletons release SDF-1 internal recruitment MSC
Activity.Simple nano-particle modified more micropore cytoskeletons are without apparent MSCs chemotactic activity as the result is shown.Show from receiving
It is more than 2 weeks that the SDF-1 of more micropore cytoskeletons release of rice grain modification can raise MSCs in vivo.Pass through release
It may be the better solution that seed cell source is regenerated for tissue repair that SDF-1, which raises MSC,.
Researches show that nano-particle modified more micropore cytoskeletons to release for external MSCs migration experiment and internal MSCs recruitment
The SDF-1 put has strong chemotactic ability, and the nano-particle modified more micropore cytoskeletons for carrying SDF-1 can be in vivo
Self MSCs is raised for a long time, obtain superior chemotactic and raises the activity of self seed cell.And continued by BMP-2
Release can provide more favorable extracellular microenvironment for bone tissue reparation.
For a further understanding of the present invention, below in conjunction with the embodiment of the present invention, to the technical side in the embodiment of the present invention
Case is clearly and completely described, it is clear that and described embodiments are only a part of the embodiments of the present invention, rather than all
Embodiment.Based on the embodiments of the present invention, those of ordinary skill in the art institute without making creative work
The every other embodiment obtained, shall fall within the protection scope of the present invention.
Unless otherwise specified, reagent involved in the embodiment of the present invention is commercial product, can pass through business canal
Road purchase obtains.Water used is deionized water.The chitosan oligosaccharide/heparin nano particle use application No. is
201510235782.1 Chinese patent disclosed in method preparation.GraphPad Prism version is all made of in this research
6 (GraphPad Software, La Jolla, CA, USA) statistics softwares are analyzed, and all results think value < 0.05 P
Difference have statistical significance.The result of all measurement datas all uses mean ± standard deviation to indicate.Two groups and multiple groups metering money
Material is analyzed using Student ' s-test and One-way ANOVA.
Embodiment 1, nano-particle modified more micropore cytoskeletons
1, the more micropore cell scaffold materials of gelatine-chitosan-agarose are prepared using chemical crosslinking freezing gel method.
Use gelatin as substrate, is crosslinked using 5% (v/v) glutaraldehyde as chemical cross-linking agent and chitosan, and agar
Sugar has the property from gel at low temperature, and triplicity prepares more micropore cytoskeletons.
(1) acetum of 5ml 1% (v/v) is added in 50ml centrifuge tube, electronic balance weighing 100mg chitosan will
It is added in above-mentioned acetum, fullys shake, makes it completely dissolved.
(2) it after chitosan is dissolved completely in acetum, weighs 100mg gelatin powder and is added thereto, protected in adition process
Hold centrifuge tube shake.40 DEG C of freeze-day with constant temperature of oven are placed in after shaking up to sterilize oven 30 minutes.Making chitosan and gelatin, it is sufficiently mixed
It is even and dissolve.
(3) it weighs quantitative agarose powder (200mg) and is placed in 5ml distilled water (ddH2O), micro-wave oven is heated to it and fills
Divide dissolution (solution becomes clear and shows sufficiently to dissolve).
(4) it after agarose solution being cooled to 50-60 DEG C at room temperature, is added in chitosan-gelatin solution and fullys shake
It mixes, (v/v) glutaraldehyde solution of 0.5ml 5% is added dropwise in blending process for being chemically crosslinked.After mixing immediately by solution
It pours into 5ml plastic injector.
(5) it is placed in be incubated for 16 hours under -14 DEG C of temperature (cryostat) and prepares freezing gel.Later, by freezing gel
It thaws in the deionized water of room temperature, and is taken out from plastic injector mold and prepare more micropore cytoskeletons.To be mostly micro-
Hole cytoskeleton is impregnated and is equipped in the beaker of 150ml deionized water, and low speed is washed overnight on the oscillator to rinse crosslinking
The unreacted aldehyde radical of agent.More micropore cytoskeletons are dried at room temperature for study its physics and chemical behavior and biofacies
The inspection of capacitive.
(6) scanning electron microscope characterizes more micropore cytoskeletons, SEM the result is shown in Figure 1.
Macro geometry (Figure 1A) visible its of more micropore cytoskeletons is semi-transparent, flaxen partially brittle solid
Material does not have significant change through 60 DEG C of basic configurations after sterile oven dried 2 hours and mechanical performance, and through PBS buffer solution
Brittleness reduces after infiltration, and elasticity increases.It is in micropore shape that the section naked eyes of more micropore cell scaffold materials are slightly visible.Under SEM
Microstructure shows (Figure 1B) that the interconnection that the microcosmic section of more micropore cytoskeletons is about 100 μm by mean size under Electronic Speculum is micro-
The irregular honeycomb appearance structure (Figure 1B) that hole and thin-walled are constituted.From microscopic appearance, more micropore cytoskeletons are shown carefully
The desired characteristics of born of the same parents' bracket, three-dimensional, microporous interconnection structure.
To more micropore cytoskeleton porositys, swelling ratio, liquid flow rate and external degradation rate are measured, as a result
Show that more micropore cytoskeleton porositys are 82%, swelling ratio 15.03,10 mlmin of liquid flow rate >-1, show more micropores
Cytoskeleton all has high porosity, swelling ratio and liquid flow rate, is suitable for cell adherence, infiltration and proliferation.
2, the combination of chitosan oligosaccharide/heparin nano particle and more micropore cell scaffold materials
(1) 5mg chitosan oligosaccharide/heparin nano particle is accurately weighed into centrifuge tube, and distilled water dissolution, acetic acid titrates nanometer
Particle suspension liquid.
(2) from original more micropore cytoskeleton samples cut into fixed size it is cylindric (diameter: 13mm, thickness:
2mm), and by assay balance it is weighed and is recorded.Ultrasonic rinsing 5 minutes, nano granule suspension centrifuge tube is added
In.
(3) centrifuge tube is placed in 37 DEG C of thermostatic water bath vibrators (60rpm), physics self assembly makes nano particle and more
Gelatin mutually adsorbs on micropore cytoskeleton, is further processed after 6 hours.
(4) 5% glutaraldehyde is added, is crosslinked nano particle further with more micropore cytoskeletons, 37 DEG C of reactions
20min.PBS buffer solution is cleaned 5 minutes × 3 times.
(5) it after the more micropore cell scaffold materials for combining nano particle being carried out freeze-drying process, is received to combining
The material of rice grain is weighed.More micropore cytoskeletons is of poor quality to calculate nanometer before and after measuring in conjunction with nano particle
The binding capacity of particle.
(6) the combination situation of scanning electron microscopic observation more micropore cytoskeletons and nano particle, scanning electron microscope
(Hitachi S-3400N, Japan) obtains image (Fig. 2).
The results show that more micropore cytoskeletons can be modified by chitosan oligosaccharide/heparin nano particle (CSO/H NP), nanometer
Particle binding capacity reaches 17.52 ± 4.16 μ gmg-1, show that the ability of more micropore cytoskeleton combination nano particles is stronger.
Embodiment 2, cell proliferation test evaluate the cytotoxicity (CCK-8 of nano-particle modified more micropore cytoskeletons
Method)
Cell Counting Kit-8 (CCK-8 kit) is detection cell Proliferation, cell survival and cytotoxicity
Kit is a kind of extensive use fast high-sensitive degree detection kit based on WST-8, is the alternative of mtt assay, examination
It can be by more Intramitochondrial in the presence of electronics coupled reagent using water-soluble tetrazolium salts-WST-8 in agent box
Dehydrogenase reduction generates orange-yellow formazan (Fig. 3).The more cell Proliferation the faster, then color is deeper；Cytotoxicity is got over
Greatly, then color is more shallow.For same cell, the depth and cell number of color are in good linear relationship.Concrete operations are such as
Under:
(1) Nature enemy cell line: the cell line of proliferation experiment to be detected uses the pure cell culture medium without serum " hungry
Starve " after processing 6 hours, kind plate to 24 porocyte culture plates.
(2) inoculating cell: the culture medium 3ml containing serum will be added to neutralize, and gently MSCs 1ml trypsin digestion
Piping and druming counts its de- wall with cell counter, 24 porocyte culture plates, 5000, every hole MSCs after cell is resuspended,
Complete medium is added to every 400 μ l of hole, 5 repeating holes of every group of setting, totally three groups (blank group, gelatine-chitosans-agarose
More micropore cytoskeleton groups (bracket group), nano-particle modified more micropore cytoskeleton groups (nanometer-bracket group)), setting 0,
24,48,72,96,120 hours time intervals (being arranged in 6 24 porocyte culture plates).
(3) cell culture: 37 DEG C, 5%CO2Cell incubator culture.It is adherent to cultivate second day group of cells, if
It is set to 0 hour.40 μ L CCK-8 solution are added to every hole in 24 orifice plates when taking 0, this step is it is noted that avoid generating bubble, gas
It steeps oneself-meeting Effect of Refraction O.D value.
(4) culture plate is incubated in incubator 2 hours (37 DEG C, 5%CO2Under conditions of).
(5) it is worth based on measuring absorbance (the OD value) at 450nm with microplate reader.
(6) packet transaction: in the cell (Fig. 4) of remaining 5 piece of 24 orifice plate merging customization, three groups do not exist together respectively
Reason: A group, blank group are without any processing；B group, more micropore cytoskeleton groups (bracket group), cell is built into unbonded nanometer
More micropore cell scaffold materials of particle；C group, nano-particle modified more micropore cytoskeleton groups (nanometer-bracket group) are small
The nano-particle modified more micropore cell scaffold materials of indoor merging.
(7) measured absorbance value by 0 hour same steps at the 24th, 48,72,96,120 hour, and be incubated for every time when
Between it is consistent with 0 hour time point, be 2min.
(8) draw three groups of cell growth curve: abscissa is each time point, and ordinate is absorbance value or lives thin
Born of the same parents' relative populations.
Nano-particle modified more micropore cell scaffold materials are by by bracket material to the assessment of the proliferative effect of MSCs
Material is incubated for altogether with MSCs to assess (Fig. 4).During incubation, nano-particle modified more micropore cell scaffold materials are immersed in
In culture medium, but do not contacted directly with cell.In order to avoid part MSCs is attached to material during being incubated for, final result is influenced
Accuracy.Three groups of MSCs are cultivated 5 days respectively, and group of cells growth curve is as shown in Figure 5.
The results show that the growth rate of MSCs is on day 1 with the 2nd day without significant difference in 3 groups.At the 3-5 days, receive
Rice modification more micropore cytoskeleton groups (nanometer-bracket group) and control group between proliferative capacity there are significant difference (p <
0.05), but the OD value of two groups of cells rose at last 3 days in alternating.And more micropore cytoskeleton groups (bracket group) and control group
Between OD value only have significant difference on day 3, and generally, the proliferative capacity of MSC does not have significant difference in two groups.Table
Bright nano-modified bracket or the bracket not being modified show do not have significant cytotoxicity to MSCs, to the proliferation shadow of MSCs
Sound is not significant.
The cell attachment evaluation of embodiment 3, nano-particle modified more micropore cytoskeletons
Nano-particle modified more micropore cytoskeletons are used for the regenerated cytoskeleton of tissue repair as a kind of, most
An important function can exactly be adhered to by stem cell.Cell attachment after can further progress proliferation and differentiation, and
Under the action of cell factor, promote tissue repair regeneration.In order to assess the cell of nano-particle modified more micropore cytoskeletons
The GFP MSCs marked is inoculated on bracket, is then incubated for after a certain period of time, sees under inverted fluorescence microscope by Adhesion property
Examine the form and adhesion condition of cell.Concrete operations are as follows:
(1) Nature enemy cell line: the GFP of convergence degree 80% or so is taken to mark MSCs.It is " hungry with not serum-containing media
Starve " processing 6-8 hours.
It (2) will be in sterilization and withering timbering material 24 porocyte culture plates of merging.
(3) inoculating cell: by MSCs with after 1ml trypsin digestion 1 minute, adding the culture medium 3ml containing serum to neutralize,
And gently blow and beat, make its de- wall.After cell is resuspended, counted with cell counter.The MSCs for taking 10000 GFP to mark is slow
It is slow to instill material surface, it is gradually immersed into bracket to it.Every hole adds 400 μ l of complete medium.
(4) then by the bracket for being vaccinated with GFP label MSCs in 24 orifice plates, it is embedded in 37 DEG C of moist 5%CO 2
It is incubated for 2 days in incubator.The form of the cell density of bracket and the cell of attachment is attached to by assessment come qualitative evaluation material
Cell can adhesion.As a result see Fig. 6.
It is attached to the high density MSC of bracket as the result is shown, the MSCs of GFP label can be attached to material as seen from the figure
In surface and micropore, star-shaped, shuttle shape or mostly prominent shape is unfolded in cell, and deducibility cell is in healthy form, and density is higher.
The result shows that nano-modified more micropore cytoskeletons are the good steel frameworks of MSCs, there is excellent cell compatibility.
Embodiment 4, cell factor are in conjunction with more micropore cytoskeleton nano surface particles and release in vitro
(1) BMP-2 or SDF-1 are diluted and is configured to various concentration cell factor solution.It will be nano-modified mostly micro-
Hole cytoskeleton is totally submerged in cell factor suspension, and 37 DEG C of thermostatic water bath vibrator (60rpm) 6h load it sufficiently.
After removing nano-modified more micropore cytoskeletons.By cell factor ELISA kit measure in solution remaining cell because
Sub- content, and computational load efficiency.Formula is as follows:
Encapsulation rate=(A-B)/A × 100%
Carrying drug ratio=(A-B)/C × 100%
In formula, A is cell factor gross mass；B is residual cell after the completion of load because of protonatomic mass；C is nano particle matter
Amount.
(2) the external release test of cell factor
1. each group is loaded the nano-modified more micropore cytoskeletons of cell factor to be placed in 10mlPBS buffer, it is placed in
37 DEG C, in 60rpm constant water bath box, concussion incubation is carried out in gnotobasis；
2. after being incubated for the scheduled time, nano-modified more micropore cytoskeletons being removed from centrifuge tube and are placed in newly
In 10mlPBS buffer.PBS sample originally respectively samples -20 DEG C of freezen protectives after 1ml, temporally with grouping to sample into
Line flag；
3. being hereafter sampled according to the time point of setting and updating incubation liquid.It is sequentially placed into the sample cell of number,
Freezen protective；
4. the sampling period is 15 days.All samples take out from -20 DEG C of refrigerators, carry out by ELISA detection kit specification
Cell factor quantitative analysis calculates the Cumulative release amount of cell factor.
(3) cell factor ELISA test experience
Rewarming is carried out at room temperature using preceding be placed in all reagents and sample.Washing buffer need to be complete to crystalline lens in bottle
It is used after the dilution proportion with deionized water by 1:25 (vol:vol) again after fully dissolved.
1. illustrating to prepare all reagents, working stamndard and sample to be tested according to previous section.It is extra to remove from sheet frame
Microwell plate, they are put back in the aluminium foil bag equipped with desiccant pack, is then resealed.
2. 100 μ LAssayDiluent RD1-55 are added into each hole.
3. 100 μ L standard items, reference substance or sample to be tested is added in every hole, sealing is covered with the adhesive tape in kit.Room temperature
Under be incubated for 2 hours in the horizontal rail microplate oscillator that revolving speed is set as 500 ± 50r/min.
4. sucking the liquid in each hole, and each hole is filled using the washing buffer (400 μ L) after dilution and is washed
It washs, then sucks washing buffer completely, repeat the process three times, wash four times altogether.It must be completely removed in each step
Otherwise washing buffer will affect the accuracy of result.After last time is washed, any remaining washing is removed by suction
Buffer.Overturning orifice plate sucks residual liquid on clean paper handkerchief.
5. 200 μ L human cell factor conjugates are added into each hole.Adhesive tape is covered again.It is set at room temperature in revolving speed
To be incubated for 2 hours in the horizontal rail microplate oscillator of 500 ± 50r/min.
6. as step 4. in repeat aspiration/washing four times.
7. 200 μ L substrate solutions are added in every hole.It is being protected from light incubation 30 minutes at room temperature.
8. 50 μ L terminate liquids are added in every hole.Color in hole should become yellow from blue.If the color in hole is green
Or color change is uneven, can beat plate gently to ensure to be thoroughly mixed.
9. absorbance measurement: using the microplate reader for being set as 450nm, the absorbance in each hole is determined in 30 minutes
(optical density, OD) value.Tuning wavelength is set as 540nm or 570nm.According to preset standard concentration and measurement
Standard concentration draw standard curve, further according to the OD value of standard curve and sample to be tested calculate sample to be tested in cell because
Sub- concentration.
The cell factor load efficiency of table 1CSO/H nano-particle modified more micropore cytoskeletons
| Group* | 2μg/mg | 1μg/mg | 500ng/mg | 250ng/mg |
| SDF-1EE (%) | 89.54±1.39 | 94.37±1.48 | 94.41±1.37 | 93.53±1.87 |
| BMP-2EE (%) | 82.73±2.95 | 92.67±1.82 | 92.85±1.52 | 93.78±1.25 |
* every mg nano particle loads 2 μ g, 1 μ g, 500ng and 250ng cell factor respectively
The cell factor load efficiency of nano-particle modified more micropore cytoskeletons as the result is shown of table 1.Nano particle
More micropore cytoskeletons SDF-1 and BMP-2 load efficiency with higher of modification (more than 80%).The physiology of cell factor
Concentration is far below 100ng/ml.Therefore, cell factor load capacity is sufficient for the needs of organizational project.
The In-vitro release curves for loading the cell factor (SDF-1 and BMP-2) in nano-particle modified bracket are shown in
In Fig. 7.The nano-particle modified bracket of SDF-1 with load shows typical initial burst in preceding 3 detection times point
Property release.It shows to mitigate release mode after 5 days.In contrast, the release profiles of BMP-2 are shown similar to medicine for power
Zero-order release profile in, whole releasing trend are more stable.However, being mounted with the nano-particle modified of SDF-1 and BMP-2
More micropore cytoskeletons effectively realize the sustained release more than 15 days.Cell factor is carried on nano-particle modified
After more micropore cytoskeletons, the bioactivity of the cell factor of sustained release can be kept for a long time.
The chemotactic activity of the SDF-1 of the nano-particle modified more micropore cytoskeletons release of embodiment 5, assessment
(Transwell method)
Nano-particle modified more micropore cytoskeletons release is studied using the cell Transwell of Corning company
The chemotactic activity of SDF-1, the bottom of cell are polycarbonate membranes, are reflected by counting the cell quantity across cell bottom
The transfer ability of cell.The small indoor referred to as upper chamber of Transwell, upper chamber are added the cell suspension counted, cell are put into specially
With culture plate, culture plate is interior to be known as lower room, and lower room is placed in different intervention factors according to experiment purpose, can be to the cell of upper chamber
Generate corresponding biological effect.Polycarbonate membrane has the micropore of 8 μm of sizes, and cell can be migrated through micropore to polycarbonate membrane
Lower surface, judge the intervention factor to the chemotactic ability (Fig. 8) of cell by counting the cell number below polycarbonate wave.
The specific method is as follows:
(1) Nature enemy MSCs: the cell line wait test carries out after being handled 6 hours with not serum-containing media " starvation "
Migration experiment.
(2) 200 μ l are separately added into the upper and lower room transwell respectively, 600 μ l serum free mediums, 37 DEG C, 5%CO2
After cell incubator is incubated for 30min, inhales and abandon culture medium.
(3) logarithmic phase MSCs 1ml trypsin digestion is taken, 10% serum-concentration culture medium of 3ml is added and neutralizes, and is light
Featheriness is beaten, its de- wall is made.It draws 1ml cell suspension to manage to 1.5mlEP, 800rpm is centrifuged 5min, adds 1 × PBS solution of 1ml
It is centrifuged within 800r/ minutes after resuspension 3min again, repetition PBS solution washing cell is primary, and 1ml is added and contains 2% serum free culture system base weight
It is outstanding, after counting cell, adjustment cell density to 2.5 × 105A/ml.
(4) upper chamber adds 200 μ l cell suspensions, and lower room adds 600 μ l, 2% serum-concentration culture medium, and point three groups of processing: A
Nano-particle modified more micropore cytoskeletons of unsupported cell factor are added in group；Individual SDF-1 is added in B group；C group adds
Enter to load nano-particle modified more micropore cytoskeletons of SDF-1；
(5) 37 DEG C, 5%CO2Cell incubator be incubated for predetermined point of time (12 hours, 24 hours, 48 hours with, it is 72 small
When).
(6) 37 DEG C were incubated for the scheduled time, reached predetermined point of time and took out cell, sucked small indoor culture medium, used
Cotton swab wipes upper chamber face away and does not pass through cell, after the cleaning of 1 × PBS solution, fixes 30min with 5% glutaraldehyde, and PBS is washed twice,
0.1% violet staining 30min, 1 × PBS solution rinsing, dries.
(7) inverted microscope, which is taken pictures, counts the cell number of polycarbonate back of the membrane migration.Random counter centre and surrounding 5
The cell number in a visual field.
As shown in the result of Fig. 9: A group is significantly lower than two groups of B, C in the MSCs quantity that four time points migrate.Show
Simple nano-particle modified more micropore cytoskeletons do not have apparent chemotaxis to MSCs, influence cell transmembrane quantity
Principal element be chemotaxis of the SDF-1 to MSCs, therefore transmembrane cell quantity is significantly lower than other two groups.And B group and C
Just there is obvious statistical difference (p < 0.01, Fig. 9), table after being incubated for 48 hours in quantity between group across the MSC of cross-film
The time that the SDF-1 of bright C group plays chemotactic activity is considerably longer than B group.Should the experimental results showed that, be carried on nano-particle modified
More micropore cytoskeletons after, SDF-1 can keep its bioactivity for a long time, persistently play chemotactic stem cell effect.
Embodiment 6, osteogenic induction experiment evaluate the BMP-2 skeletonization of nano-particle modified more micropore cytoskeletons release
Induced activity
The induction of the BMP-2 of nano-particle modified more micropore cytoskeletons release is studied using osteogenic induction experiment
The osteogenic activity of MSCs.4 groupings: A group, nano-particle modified more micropore cytoskeleton groups of blank are set；B group: BMP-
2 groups, the simple BMP-2 that is added is induced；C group: nano-particle modified more micropore Xi Baozhijia &BMP-2 groups, merging are loaded with
Nano-particle modified more micropore cytoskeletons of BMP-2 are induced；D group: positive controls, using osteogenic induction liquid into
Row induction.Pass through the activity for the BMP-2 that the bracket that the detection of osteogenic induction measuring method is modified from NP discharges.Concrete operations are as follows:
(1) C57BL/6 Bone Marrow Mesenchymal Stem Cells are placed in 37 DEG C, 5%CO2Incubator in cultivate.
(2) it when cell fusion degree reaches 80-90%, is digested with 0.25%Trypsin-0.04%EDTA.
(3) MSC (4 × 10 that will be digested4) be inoculated in each hole of 24 orifice plates with normal incubation medium.24
Orifice plate is coated with 0.1% gelatin in advance, and 400 μ l complete mediums are added in every hole.
(4) cell is placed in 37 DEG C, 5%CO2Incubator in cultivated.
(5) when cell fusion degree reaches 60%-70%, careful siphons away complete medium in hole.Respectively according to pre-
If 4 groupings intervened, two pack support of A, C be placed in customization cell in, avoid directly contacting (Figure 10) with MSCs.
(6) it used half amount of A, B, C group complete medium instead every 3 days and changes liquid, D group is complete using MSCs Osteoinductive differentiation
Full culture medium half, which is measured, changes liquid (needing to be preheated to 37 DEG C before use).
(7) it after being incubated for scheduled time (7 days, 14 days and 21 days) in 2 incubator of 5%CO humidified at 37 DEG C, uses
ALP microwell plate test kit (Bioengineering Research Institute is built up in Nanjing) measures the alkaline phosphatase activities in each hole.Every hole
In sample with 0.1% Alizarin red staining and use microscope imaging.The result is shown in Figure 11.
Figure 11 A in the group of A group (nanometer-bracket group) for lacking induced medium the results show that observe than at other
Much lower ALP activity in group, it means that pure bracket or the nano particle from bracket have no induction MSCs early stage skeletonization
Activity.On the contrary, other group (B group, BMP-2 groups；C group is loaded with nanometer-bracket group of BMP-2；D group, positive controls) display
Apparent ALP positive findings out (compared with A group, p < 0.05).And it is thin to be exposed to C group (nanometer-bracket group for being loaded with BMP-2)
The ALP activity of born of the same parents was shown and the similar ALP of B group (BMP-2 group) activity at the 7th day.However, C group ALP activity increases speed
Degree is very fast, and the 14th day and the 21st day, ALP activity was apparently higher than B group (p < 0.01), and (positive close to D group in 14 days
Control group) (p < 0.05).It can be seen that the BMP-2 sustained release that nano-particle modified more micropore cytoskeletons load may
It is continuous to play osteoinductive activity, and the ALP activity in the group of BMP-2 is loaded finally at the 21st day more than D group, but gap
Not significant (p=0.09).
By alizarin red to the degree of cell dyeing further qualitative evaluation doped calcium and calcium tubercle.7 after being inoculated with MSC
It, 14 days and 21 days it is as shown in Figure 11 B to cell Alizarin red staining result, B group (BMP-2 group), C group (are loaded with receiving for BMP-2
Rice-bracket group) and the apparent alizarin red positive staining of D group (positive controls) display, and A group (nanometer-bracket group) cell exists
Almost without positive staining after being persistently incubated for 21 days.The result shows that bracket or nano particle do not have promotion MSCs Osteoblast Differentiation
Apparent activity.Compared with B group, more significant positive staining result is also showed that from C group the 14th day and the 21st day.It is heavier
It wants, most deep Alizarin red staining result is presented after being incubated for 21 days in C group in four groups, it means that independent relative to B group
For BMP-2 intervenes, the nano-particle modified more micropore cytoskeletons of C group and cell factor play significantly in self-bone grafting
Synergy.
Embodiment 7, IVIS assess the internal chemotactic activity of nano-particle modified more micropore cytoskeletons release SDF-1
The SDF- of nano-particle modified more micropore cytoskeletons release is studied using small animal living body fluoroscopic imaging systems
1 internal recruitment MSCs activity.Two groups: A group of A, B is set in the nano-particle modified more micropores for being subcutaneously implanted load SDF-1
Cytoskeleton；B group is subcutaneously implanted nano-particle modified more micropore cytoskeletons (control group) of unsupported cell factor.Tool
Gymnastics is made as follows:
(1) male 7 week old BALB/c nude mices (Hunan Si Laike reaches the offer of experimental animal Co., Ltd unexpectedly) is taken, according to setting
The grouping set receives material implant surgery respectively: after with amobarbital (50mg/kg) anesthesia, tweezers lift nude mice back skin
Skin, eye scissors laterally cut off skin, incision length about 1cm.Tweezers lift notch skin edge, eye scissors blunt separation subcutaneous fascia
To muscle layer.Continue blunt separation and expands subcutaneous space, after grouping requirement implantation material, the continuous skin suture notch of 1# silk thread.
(2) cell is resuspended: the GFP of convergence degree 80% or so being taken to mark MSCs.It is handled with not serum-containing media " starvation "
After 6-8 hours.Culture medium is sucked, 2mlPBS buffer rinses one time.1ml pancreatin digestive juice is added, 37 DEG C are incubated for 2 minutes, add
Enter in 3ml complete medium and digestive juice.It is centrifuged 3 minutes within 800r/ minutes after cell count.GFP is marked with physiological saline
MSCs is diluted to 1 × 107A/ml.
(3) after predetermined point of time (being grouped within 1 day, 3 days, 7 days, 10 days, 15 days and 18 days), by 1 × 10 in 100 μ l6
A GFP-MSC is injected into tail vein.48 hours after cell infusion, after sevoflurane gas anesthesia, it is imaged by small animal living body
Mouse is observed under system spectrum (IVIS Spectrum, PerkinElmer) and is imaged, it is strong by the green fluorescence of implantation position
Spend the recruitment effect in vivo to MSCs of qualitative evaluation SDF-1.Experimental result is as shown in figure 12.
The results show that A group back of mice excites relatively high fluorescent brightness, and fluorescence is the 15th in initial 10 days
It becomes weaker, then final to disappear.On the contrary, B group is at all time points all without display fluorescence, it can be seen that simple receives
More micropore cytoskeletons of rice grain modification are without apparent MSCs chemotactic activity.This is the result shows that from nano-particle modified more
It is more than 2 weeks that the SDF-1 of micropore cytoskeleton release can raise MSCs in vivo.Raising MSC by the SDF-1 of release may
It is the better solution that seed cell source is regenerated for tissue repair.
Claims (7)
1. a kind of osseous tissue renovating material, which is characterized in that including nano-particle modified more micropore cytoskeletons and cell because
Son.
2. osseous tissue renovating material according to claim 1, which is characterized in that nano-particle modified more micropores are thin
Born of the same parents' bracket is by nano particle that is positively charged and being chemically crosslinked and the more micropore cytoskeletons of gelatine-chitosan-agarose
Material is combined into.
3. osseous tissue renovating material according to claim 2, which is characterized in that the nano particle is chitosan oligosaccharide/heparin
Nano particle.
4. osseous tissue renovating material according to claim 2, which is characterized in that the gelatine-chitosan-agarose is mostly micro-
Hole cell scaffold material is prepared by chitosan, gelatin and agarose using chemical crosslinking freezing gel method, the gelatin-shell
Glycan-agar sugared content is 4~5wt%, and the mass ratio of the chitosan, gelatin and agarose is 1:1:(2~3).
5. osseous tissue renovating material according to claim 4, which is characterized in that the chemical crosslinking freezing gel method is specific
Addition gelatin mixes well dissolution after being dissolved completely in acetum for chitosan, and agarose solution is added and mixes, and mixed
5% (v/v) glutaraldehyde solution is added dropwise during even to be crosslinked, is subsequently placed at -14 DEG C and is incubated for the freezing of preparation in 16 hours
Gel, thaw at RT prepare more micropore cytoskeletons.
6. osseous tissue renovating material according to claim 2, which is characterized in that nano-particle modified more micropores are thin
The preparation method of born of the same parents' bracket is to obtain nano granule suspension after nano particle is resuspended in water, and gelatine-chitosan-agarose is mostly micro-
Hole cell scaffold material, which is added in nano granule suspension, shakes 6 hours for 37 DEG C, and 5% 37 DEG C of reaction 20min of glutaraldehyde are added,
It is freeze-dried after cleaning.
7. osseous tissue renovating material according to claim 1, which is characterized in that the cell factor be SDF-1 and/or
BMP-2。
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| CN101869724A (en) * | 2009-04-27 | 2010-10-27 | 裴国献 | Bone repair stent material capable of realizing controlled-release of traditional Chinese medicine and preparation method thereof |
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|---|---|---|---|---|
| CN101869724A (en) * | 2009-04-27 | 2010-10-27 | 裴国献 | Bone repair stent material capable of realizing controlled-release of traditional Chinese medicine and preparation method thereof |
| CN102690436A (en) * | 2012-06-11 | 2012-09-26 | 北京大学 | Active microspheres capable of directionally regulating and controlling chondrocyte accumulation and preparation method of active microspheres |
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