CN107261204A - A kind of application of method and its bone tissue engineering scaffold modification of the area light graft growth factor - Google Patents

A kind of application of method and its bone tissue engineering scaffold modification of the area light graft growth factor Download PDF

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CN107261204A
CN107261204A CN201710335943.3A CN201710335943A CN107261204A CN 107261204 A CN107261204 A CN 107261204A CN 201710335943 A CN201710335943 A CN 201710335943A CN 107261204 A CN107261204 A CN 107261204A
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bone tissue
growth factor
light
dimensional material
igf
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关燕清
汪慧敏
陈吾雅
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South China Normal University
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South China Normal University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/16—Macromolecular materials obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A61L2300/414—Growth factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/18—Modification of implant surfaces in order to improve biocompatibility, cell growth, fixation of biomolecules, e.g. plasma treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants

Abstract

The invention discloses a kind of method using the ultraviolet interference of light/diffraction characteristic area light graft growth factor and its application of bone tissue engineering scaffold modification.The present invention can be interfered using ultraviolet light or the characteristics of diffraction, under the action of uv light, by insulin-like growth factor 1（IGF‑1）Regionality is grafted to bone tissue engineer two-dimensional material surface, and structure obtains regional photo-grafting support.The timbering material has the effect of obvious regional induction differentiation to bone mescenchymal stem cell, so as to reach the purpose of directional induction.And the present invention provides a kind of new approaches for new timbering material exploitation, deepens the understanding to cell differentiation, while exploitation is conducive to the timbering material that field of tissue engineering technology is used.

Description

Method and its bone tissue engineering scaffold modification of a kind of area light graft growth factor Using
Technical field
The invention belongs to biological medicine Material Field.The ultraviolet interference of light/diffraction characteristic area is utilized more particularly, to one kind The application of method and its bone tissue engineering scaffold modification of domain photo-grafting growth factor.
Background technology
Clinically, the treatment of bone injury is one of orthopaedics problem urgently to be resolved hurrily all the time, in recent years bone tissue engineer Develop rapidly for people solve bone injury treatment provide a kind of new thinking.Traditional bone tissue engineer refers to, The Gegenbaur's cell of culture is transferred to a kind of technical method through in vitro culture formation mineralising bone on biologic bracket material.Its is basic Principle is, using the Gegenbaur's cell cultivated in vitro as seed cell, to be planted with good biocompatibility and biodegradation Property three-dimensional stent material on, then cell/scaffold complex implanted or continues to cultivate in vitro, cell by breeding, Differentiation, extracellular matrix secretion formation mineralising bone tissue, while biological support structure is gradually degraded, absorbed, are repaiied so as to reach Multiple bone injury, the purpose for rebuilding bone function.The harm that the importance and bone related disease of bone tissue are brought, accelerates bone tissue work The development of journey.Under this overall background, bone tissue engineer turns into and repaiied because of the great potential with treatment bone associated injury or disease The method in the new forward position of multiple affected organ.
Typically, the research of bone tissue engineer contains three big elements：Timbering material, seed cell and growth factor. Wherein, timbering material is as the framework of bone tissue regeneration, and its characteristic directly affects the biological characteristics of seed cell, influence cell Existence, migration, propagation and metabolic function, therefore it is one of key element in bone tissue engineer.By taking timbering material as an example, This laboratory is prepared for this kind of biomaterial of PAAm-IGF-1/TNF- α, and successfully induces BMSC after inoculation BMSC on the support To hepatocyte differentiation.But this kind induction is single, there is limitation.And bone tissue is different with function by many forms Identical cell is combined into a certain way, differentiates no Regional Differentiation by BMSC and single cell is to can not meet The requirement hindered at present for the treatment assessment of loss.There is researcher to attempt growth factor compartmentalization being grafted on support using grating On, but be that the requirement to grating is too high the drawbacks of this kind of method, and a kind of grating can only be grafted a kind of support of compartmentalization, It certainly will be the waste to resource.
In terms of seed cell, different growth factor-induced stem cell differentiation, it is to grind at present to form various types of cells Study carefully one of more active field.Mescenchymal stem cell (MSC, mesenchymal stem cells) is the weight of stem cell line Member is wanted, from the mesoderm and ectoderm of mesoderm growing early stage, belongs to multipotential stem cell, MSC initially has found in marrow, because of it With multi-lineage potential, hematopoiesis support and promote stem cell implantation, immunoregulation and be increasingly subject to the features such as self-replacation The concern of people.Under mescenchymal stem cell in vivo or in vitro specific inductive condition, fat, bone, cartilage, flesh can be divided into The Various Tissues cell such as meat, tendon, ligament, nerve, liver, cardiac muscle, endothelium, still has many after continuous passage culture and freezen protective To differentiation potential, injuries of tissues and organs reparation caused by aging and lesion can be used for as preferable seed cell.
Research shows, on the problem of support is modified, and purposefully adds different while cell is cultivated on support Growth factor, can effectively induce it to be divided into different cell types.In tradition research mode, growth factor is with trip From form add in extracellular environment, but their effect can not keep longer time.Growth factor is fixed on Its action effect is extended on support, can't say that it is wrong a kind of brand-new method.
The content of the invention
The technical problem to be solved in the present invention is the defect and deficiency for overcoming above-mentioned prior art, can be occurred using ultraviolet light The characteristics of interference or diffraction, under the action of uv light, insulin-like growth factor 1 (IGF-1) regionality is grafted to polyphenyl Ethene (PS) two-dimensional material surface, successfully constructs a kind of new bone tissue engineering scaffold.The rat bone cultivated simultaneously thereon Mescenchymal stem cell (BMSCs), studies the effect that the support grows and broken up to rat bone mescenchymal stem cell (BMSCs), knot Fruit finds significantly regional differentiating phenomenon, and the timbering material has the effect of obvious regional induction differentiation to BMSCs, real The purpose of existing directional induction.
It is an object of the invention to provide a kind of side using the ultraviolet interference of light/diffraction characteristic area light graft growth factor Method.
Another object of the present invention is to provide the regional photo-grafting support that the above method is prepared.
Another object of the present invention be to provide the regional photo-grafting support as or prepare bone tissue engineering scaffold The application of aspect.
Above-mentioned purpose of the present invention is achieved through the following technical solutions：
A kind of method using the ultraviolet interference of light/diffraction characteristic area light graft growth factor, can be sent out using ultraviolet light The characteristics of raw interference or diffraction, under the action of uv light, by the grafting of insulin-like growth factor 1 (IGF-1) regionality, (light connects Branch) bone tissue engineer two-dimensional material surface is arrived, structure obtains regional photo-grafting support.
Wherein it is preferred to, the bone tissue engineer two-dimensional material is high molecular polymer class two-dimensional material.
It is highly preferred that the bone tissue engineer two-dimensional material is polystyrene (PS), PCL-PLA or PLGA.
It is highly preferred that the bone tissue engineer two-dimensional material is polystyrene (PS).
Most preferably, the bone tissue engineer two-dimensional material is 1~3cm of diameter polystyrene two-dimensional material.
Preferably, the insulin-like growth factor 1 first passes through after photoactivation photo-grafting to two-dimensional material surface, photo-grafting Condition be interfere or 220~260W uviol lamps of diffraction under handle twice, processing time is 10~30min.
Specifically, the above-mentioned method using the ultraviolet interference of light/diffraction characteristic area light graft growth factor, including following step Suddenly：
S1. photolytic activity IGF-1 is prepared：
Under the conditions of lucifuge, in the DMF/PBS solution that IGF-1 is added to the succinimide ester containing N-, in 0~4 DEG C of (ice Bath) under conditions of 40~50h of stirring reaction；After end of synthesis, with ultra-filtration centrifuge tube (Milipore Molecut II, 10KNa), Centrifugal purification；Freeze-drying is standby；
Before use, adding 50ml PBS solution dissolving, and adjust to required concentration 1ng/ μ l, obtain photolytic activity IGF-1 molten Liquid, lucifuge, Cord blood；
S2. it is modified bone tissue engineer two-dimensional material
Spontaneously dry, done under the conditions of photolytic activity IGF-1 solution, lucifuge is added dropwise to bone tissue engineer two-dimensional material surface Dry after-poppet；Dry after-poppet and be placed in optical system (optical system can realize two-slit interference or the single slit diffraction of ultraviolet light) In, 10~30min is handled under 220~260W uviol lamps；1~6min of deionized water rinsing, natural air drying；Photolytic activity is added dropwise again Spontaneously dried under the conditions of IGF-1 solution lucifuges, dry after-poppet conversion angle, be again placed in the optical system, 220~ 10~30min is handled under 260W uviol lamps, growth factor modified type support is obtained, i.e., regional photo-grafting support.
Wherein it is preferred to, N- succinimide esters in the DMF/PBS solution of the succinimide ester containing N- described in step S1 Mass concentration is 5~19 μ g/mL.
Preferably, the pH=7~8, DMF and PBS of the DMF/PBS solution of the succinimide ester containing N- described in step S1 body Product is than being 4：1.
Preferably, in step S1, IGF-1：The mass volume ratio of the DMF/PBS solution of the succinimide ester containing N- be 40~ 60μg：10~30ml.
Preferably, lucifuge condition described in step S1 can be realized by the brown vial with bottle cap, will contain N- succinimides The DMF/PBS solution of ester is added in brown vial, adds IGF-1.
Preferably, stirring reaction condition described in step S1 is 4 DEG C of (ice bath) stirring reaction 48h.
Preferably, the condition of centrifugal purification described in step S1 for 3500~4500rpm/min rotating speed under centrifugation 20~ 40min。
Preferably, the condition of centrifugal purification described in step S1 for 4000rpm/min rotating speed under centrifuge 30min.
Preferably, bone tissue engineer two-dimensional material described in step S2 is 1~3cm of diameter circular PS materials.
Preferably, the consumption of photolytic activity IGF-1 solution described in step S2 is 50~100 μ l.
Preferably, the concentration of photolytic activity IGF-1 solution described in step S2 is 1ng/ μ l, and solvent is PBS solution.
In addition, the present invention also provides a kind of region using the ultraviolet interference of light/diffraction characteristic area light graft growth factor Property photo-grafting support, its preparation method as described above, the regional photo-grafting support, and its as or prepare bone tissue work Application in terms of engineering support, all within protection scope of the present invention.
Characteristic --- two-slit interference and single slit diffraction of the invention according to ultraviolet light, it is intended to by the fine region of growth factor That changes is grafted on support.In physics, the two-slit interference of light and single slit diffraction phenomenon are very common phenomenons.Two row light waves It is superimposed, always strengthens in some regions when space is met, always weaken in other region, so that between there is bright dark phase Interference of the phenomenon of striped light.Always identical wave source is relevant wave source for two Vibration Conditions, and be only concerned with wave source hair The light gone out is mutually superimposed, could produce interference, occurs the striped between stable bright dark phase on screen.Light is running into barrier When, the phenomenon for deviateing straightline propagation direction and being irradiated to shadow region is called the diffraction of light.When the size of hole or barrier compares light Ripple wavelength is small, or with wavelength it is similar when, obvious diffraction phenomena could occur for light.Parallel monochromatic light, which projects one, to be had On the baffle plate of two slits, slit close proximity, the light wave of directional light can pass to slit simultaneously, and they are just into two vibration feelings Always identical wave source is referred to as relevant wave source to condition, and space of the light that they send behind plate washer is overlapped mutually, just there occurs dry Relate to phenomenon.When monochromatic light is after double slit, light and dark interference fringe is generated on screen.When the upper somewhere of screen and two slits Path length difference when being the even-multiple of half-wavelength, then the crest superposition of two train waves, trough is superimposed with trough, formation bright fringes.Work as screen When the path length difference of upper somewhere and two slits is the odd-multiple of half wavelength, it is overlapped mutually in these local crests with trough, light The amplitude of ripple cancels each other, and dark fringe occurs.Single slit diffraction is that light runs into barrier in communication process, and light wave can cut-through Thing continues a kind of phenomenon propagated.If wavelength is with seam, hole or suitable or bigger barrier size, diffraction phenomena is most bright It is aobvious.Fresnel is supplemented on the basis of Huygen's principle, gives the quantitative description on position phase and amplitude, proposes wavelet phase The concept of dry superposition.The wavelet sent by co-phasal surface AB only produces and the distance in BC faces is being turned to by AB faces to the optical path difference of P points Between.The rest may be inferred, as m=2k (k=1,2,3 ...), i.e. when m is even number, and dark line occurs in diffracted ray convergent point on screen.Screen The vibration of upper P points is exactly this half-wave zone in vibrations synthesis caused by the point, then bright spot occurs on screen, that is, bright line is presented. From above content, for two-slit interference and the condition of single slit diffraction, equipment will be easier to prepare, compared to preparing grating Say, equipment making prepared by this laboratory oneself is simple, easy to operate.Young's double-slot interference phenomenon be generally used for measurement wavelength, Measure the thickness and refractive index and the small variable of measurement of length of film；It is wide that single slit diffraction phenomenon is generally used for measuring slit The measurement of degree and filament diameter.In this experiment, we are beneficial to two-slit interference and single slit diffraction both physics of ultraviolet light Phenomenon allows directional light that the distribution of compartmentalization can be presented on support.Meanwhile, then combine the crosslinking technology of ultraviolet light by photolytic activity Growth factor is zonal to be grafted on support.
The present invention carries out Experiments of Optics simulation by computer software Matlab 2014a first, explores region photo-grafting most Suitable experiment condition, and by way of chemical modification, the photoactivation IGF-1 factors.Then, the base of optical experiment bench is being built On plinth, using ultraviolet light interference or diffraction the characteristics of, a kind of growth factor of photofixation is in PS material surfaces.Therefore, new branch It is prepared by frame.A variety of characterization methods, including Raman spectrum, immunofluorescence etc., for the sign to support.Thereafter, it is detection Its biological effect, we are grown to BMSCs by the experimental verification such as immunofluorescence, SABC support and induce differentiation Influence.As a result show：New-support is successfully constructed, and with the effect of induction stem cell regionality differentiation.Particularly, growth because Son is made the growthform of the obvious compartmentalization of BMSCs presentations by fine being grafted in the different zones of support.
Therefore, the present invention combines ultraviolet radiation graft technology and two-slit interference and the single slit diffraction characteristic of ultraviolet light, in double slit On the basis of interference and single slit diffraction, complicated irregular region is designed, is changed further to ultraviolet radiation graft technology Enter, successfully being grafted on photolytic activity growth factor I GF-1 compartmentalizations on support, successfully construct new bone tissue engineer branch Frame, New-support has the effect of obvious regional induction differentiation to BMSCs, can be divided into skeletonization to BMSC compartmentalizations Cell, so as to reach the purpose of directional induction.
The invention has the advantages that：
The present invention is using the ultraviolet interference of light and the characteristic of diffraction, using the method for region photo-grafting, successfully by this kinds of IGF-1 Insulin-like growth factor regionality is grafted to the different parts of material.A kind of new think of is provided for new timbering material exploitation Road, deepens the understanding to cell differentiation, while exploitation is conducive to the timbering material that field of tissue engineering technology is used.
Moreover, the regional photo-grafting support of the present invention has the effect of induction differentiation, support to bone mescenchymal stem cell The cell of upper different zones can break up to Gegenbaur's cell direction, so as to reach the purpose of directional induction.
Brief description of the drawings
Fig. 1 is to carry out computer simulation to ultraviolet light interference phenomena using Matlab 2014a.
Fig. 2 is to carry out computer simulation to ultraviolet light diffraction using Matlab 2014a.
Fig. 3 is the infrared detection of PS stent area photo-grafting IGF-1 after-poppets.
Fig. 4 is quantitative for the IGF-1 loadings of region photo-grafting after-poppet.
Fig. 5 is the Immunofluorescence test of PS stent area photo-grafting IGF-1 factor after-poppets.
Fig. 6 be immune-blotting method region photo-grafting support be grafted cell after differentiation from BMSC to Gegenbaur's cell.
Fig. 7 be immune-blotting method region photo-grafting support be grafted cell after differentiation from BMSC to Gegenbaur's cell.
Embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention Limit in any form.Unless stated otherwise, the reagent of the invention used, method and apparatus routinely try for the art Agent, method and apparatus.
Unless stated otherwise, agents useful for same and material of the present invention are purchased in market.
Specifically, used in following examples material, reagent and instrument are mainly as follows：
Cell line：Rat bone mescenchymal stem cell (BMSCs) is provided by medical college of Zhongshan University animal center, through this experiment Room Secondary Culture.
Main agents：IGF-1 (insulin-like growth factor), pancreatin, DMEM in high glucose culture medium, cell climbing sheet (PS materials Material), L-AA, dexamethasone, sodium β-glycerophosphate be GIBCOBRL Products；N- succinimide esters purchased from Ah Latin company；New fetal calf serum is purchased from Hangzhou Sijiqing Biological Engineering Material Co., Ltd.；24 hole polystyrene tissue cultures Substrate is Corning Corning Incorporateds of U.S. product.
Instrument：German LEO companies field emission scanning electron microscope:LEO 1530VP, Nikon microscopes, Japanese Olympus companies Optics inverted microscope, Sigma32184 high speed freezing centrifuges, Thermo CO2Incubator, Jintan City of Jiangsu Province Medical Instruments Factory's 78-1 magnetic stirring apparatus, HV-85 autoclaves, aseptic operating platform, Guangzhou Ke Qiao experimental technique equipment Co., Ltd constant temperature Water-bath etc..
The statistical analysis of following examples carries out variance analysis using spss19.0 statistical softwares, and analytic function is LSD And Duncan, P<0.05 represents significant difference.
The computer simulation of the Experiments of Optics of embodiment 1 and conditional FP tree
1st, new document is set up in Matlab 2014a softwares, and following procedure is inputted in program window, to simulate Ultraviolet light Dual-slit Interference Experiment：
Lam=250e-9；
D=1e-2；
D=0.1；
Ym=5*lam*D/d；
Xs=ym；
N=101；
Ys=linspace (- ym, ym, n)；
For i=1:n
R1=sqrt ((ys (i)-d/2) .^2+D^2)；
R2=sqrt ((ys (i)+d/2) .^2+D^2)；
Phi=2*pi* (r2-r1) ./lam；
B(i,:)=sum (4*cos (phi/2) .^2)；
end
N=255；
Br=(B/4.0) * N
subplot(1,2,1)
image(xs,ys,Br)；
colormap(gray(N))；
subplot(1,2,2)
plot(B,ys)；
>>Edit_interval=edit_lam*edit_D/ (edit_d*10000)；
Num1=str2num (get (handles.edir_lam, ' string'))；
Num2=str2num (get (handles.edit_D, ' string'))；
Num3=str2num (get (handles.edit_d, ' string'))；
Num4=num2str (num1*num2/ (num3*10000))；
set(handles.edit_interval,'string',num4)；
2nd, following procedure is inputted, to simulate ultraviolet light diffraction experiment：
A=0.001；
B=0.001；
Lmda=250e-9；
F=0.79；
Xm=0.075；
Def=0.001；
I0=1；
[x, y]=meshgrid (- xm:def:xm)；
Alpha=(pi*a*x)/(f*lmda)；
Beta=(pi*b*y)/(f*lmda)；
I=I0* (sin (alpha) .^2.*sin (beta) .^2./((alpha.^2+eps) .* (beta.^2+eps)))；
figure
imshow(I*255)
xlabel('x')；
ylabel('y')；
figure
mesh(x,y,I)
xlabel('x')；
ylabel('y')；
Zlabel (' light intensity ')；
Export image results.
3rd, result
We are successfully simulated using MatLab 2014a softwares to the two-slit interference phenomenon of ultraviolet light.When we are defeated The experiment condition entered is λ=250nm, D=10cm, during d=1cm, wherein, λ is ultraviolet wavelength, D be square hole to optical screen away from From d is the spacing of double slit, and acquired results are as shown in Figure 1.I.e. when the ultraviolet light that wavelength is 250nm entered the double slit that spacing is 1cm Afterwards, when being incident upon on the optical screen at double slit 10cm, by the center of presentation at intervals of 25 μm of light and dark striped.We select D=10cm, d=1cm are that condition carries out subsequent experimental.
Equally, the single slit diffraction phenomenon of ultraviolet light is simulated using MatLab 2014a softwares.Input λ= 250nm, D=79cm, a=1~5mm, during b=1~5mm, wherein, λ is ultraviolet wavelength, and D is square hole to the distance of optical screen, a, B is the length and width of square hole, and the ultraviolet optical diffraction simulation drawing of gained is as shown in Figure 2.I.e. when the ultraviolet light that wavelength is 250nm entered spacing Length for 1cm and it is wide be 1~5mm square hole after, when being incident upon on the optical screen at double slit 79cm, will present such as Fig. 2 institutes The bright fringes region shown.
After hot spot distribution in more above-mentioned five, we will select a=5mm, b=5mm to be that condition carries out subsequent experimental.
Embodiment 2 prepares region photo-grafting support using the ultraviolet interference of light/diffraction characteristic area light graft growth factor
1st, preparation method
(1) preparation of photolytic activity growth factor
A brown vial with bottle cap is taken, 40~60 μ g IGF-1 is added into 10~30ml contains 150~190 μ g N- ambers (pH=7~8, volume ratio is 4 to the dimethylformamide DMF/PBS of imide ester：1) in solution, 4 DEG C (ice bath, lucifuge) Under the conditions of stirring reaction 48h.After end of synthesis, respectively with ultra-filtration centrifuge tube (Milipore Molecut II, 10KNa), Under 4000rpm/min rotating speed, centrifugation 30min is freeze-dried standby with purifying azidophenyl derivative.
Before use, adding 50ml PBS solution dissolving, and adjust to required concentration 1ng/ μ l, obtain photolytic activity IGF-1 molten Liquid, lucifuge, Cord blood.
(2) modification of polystyrene (PS) two-dimensional material
The μ l of photolytic activity IGF-1 solution 50~100, lucifuge condition is added dropwise to 1~3cm of diameter circular PS timbering materials surface It is lower to spontaneously dry, obtain drying after-poppet；Dry after-poppet be placed in optical system, under 220~260W uviol lamps handle 10~ 30min.1~6min of deionized water rinsing, natural air drying.It is added dropwise under the conditions of the μ l of photolytic activity IGF-1 solution 50~100, lucifuge certainly So dry；After-poppet conversion angle is dried, is again placed in the optical system, handle 10 under 220~260W uviol lamps~ 30min, obtains growth factor modified type support, i.e. region photo-grafting support.
Wherein, described optical system can realize two-slit interference or the single slit diffraction of ultraviolet light.Specifically, the present inventor In experiment, the easy device of a kind of two-slit interference that can realize ultraviolet light or single slit diffraction phenomenon has been made by oneself.
Specifically, the device of the two-slit interference of ultraviolet light is realized：A face convex mirror is set gradually in the side of ultraviolet lamp (magnifying glass), the opaque baffle plate with two slits, and fix the part of polystyrene PS materials.
The ultraviolet light that ultraviolet lamp is sent is forming a branch of parallel ultraviolet afterwards by a face convex mirror (magnifying glass), parallel Ultraviolet light is continued through after the opaque baffle plate with two slits, is irradiated on polystyrene PS materials.The fluctuation of light beam Matter make it that the light beam by two slits is interfered with each other, and causes the bright fringes and dark striped for being shown in detecting screen.
Single slit diffraction is similarly：Single slit diffraction is that light runs into barrier in communication process, and light wave can the continuation of cut-through thing Propagate, " ten " shape striped is left on polystyrene PS materials.
2nd, the infrared spectrum detection of region photo-grafting support
Processing is dried in bare stent and the support by modification, upper machine is determined.
Fig. 3 is to respective reaction thing and the infrared spectrum of Characterization of The Products using fourier transform infrared spectroscopy.According to Fig. 3 In experimental result, spectrogram has strong absworption peak representative-OH at 3446.7；There is strong absworption peak at 1648.69, representative-C=O, Just there are these groups in growth factor I GF-1.So when this kind of insulin type photolytic activity of IGF-1 on polystyrene PS grafting materials Growth factor and polystyrene PS materials, after found by infrared detection on PS materials, with this kinds of the upper IGF-1 of control group grafting Photolytic activity growth factor is compared, and respectively has a characteristic absorption peak in 3446.7 wavelength and 1645.2 wavelength, is hydroxyl and carbonyl respectively Base, shows that this kind of growth factor of IGF-1 has been grafted on support.
The loading of the IGF-1 growth factors of embodiment 3 is quantitatively detected
1st, respectively by 0,100,200,300,400,500ng photolytic activity IGF-1 be dissolved in 3ml 95% Coomassie brilliant blue- In ethanol solution, sample OD values are detected at wavelength 540nm with ultraviolet specrophotometer, so as to draw standard curve.Meanwhile, inspection The OD values of remaining photolytic activity IGF-1 dispersion liquids after treated support are surveyed, standard curve is compared, draws IGF-1 surpluses.With adding Gross mass subtracts surplus before carrying, and produces IGF-1 loading capacities on support.Similarly, using dying method with coomassie brilliant blue, in wavelength IGF-1 loading capacity is measured at 540nm.
2nd, growth factor loading quantitative result is as shown in Figure 4.
Experimental result in Fig. 4, after this kind of insulin-like growth factor of IGF-1 on polystyrene PS grafting materials Found by UV spectrophotometer measuring, compared with control group CK, IGF-1 has 47.1% to be grafted on timbering material.Knot Fruit shows, the photolytic activity albumen of upper part can be grafted by this kind of area light grafting method.
The Immunofluorescence test region photo-grafting support of embodiment 4
1st, the preparation of SD rat bone marrow mesenchymal stem cellses Osteoinductive differentiation culture medium
The culture medium of induction SD rat bone marrow mesenchymal stem cellses Osteoblast Differentiations includes：SD rat bone marrow mesenchymal stem cellses The special hyclone 20ml of Osteoinductive differentiation basal medium 175ml, SD rat bone marrow mesenchymal stem cellses culture, glutamic acid Sodium 2ml, dual anti-2ml, ascorbic acid 400 μ l, sodium β-glycerophosphate 2ml, the μ l of dexamethasone 20.
2nd, Immunofluorescence test region support
The preparation of experiment region photo-grafting support：Optical system described in embodiment 2 is well placed as requested, system is taken The standby successful μ l of photolytic activity protein I GF-1 20 are uniformly coated on cell climbing sheet.After air-drying, cell climbing sheet is fixed on optics The screen position of part, with ultraviolet light 10 minutes.Then turn off uviol lamp, glazing activated protein IGF-1 cells will be grafted Creep plate is placed in 40mm batch cultur wares, and TBST buffer solutions are washed 3 times, 5 minutes every time, are air-dried after washing.
The IGF-1 factor modification type branch that will complete is placed in 40mm batch cultur wares, adds the closing of 2ml confining liquids 30min.Confining liquid is sucked, PBS is washed twice, the unnecessary water of branch frame peripheral is cleaned with blotting paper or toilet paper.According to IGF-1 primary antibodies Specification, primary antibody is diluted in proportion, adds the primary antibody for two kinds of albumen that 2ml TBST dilute, 4 DEG C overnight (18h or 24h). Then, cleaned 2~3 times with PBS, according to Andy FlouTM488 secondary antibody specifications dilute two kinds of fluorescence secondary antibodies, add 2ml bis- It is anti-.Notice that primary antibody is reclaimed after used.2h is incubated at room temperature, notices that lucifuge is operated.Washed with PBS after 2 times, be placed on fluorescence and show Micro- Microscopic observation is simultaneously taken pictures.
3rd, we use λ=250nm, D=10cm, and d=1cm is that condition is tested, as a result such as Fig. 5.
After synthesis photolytic activity growth factor I GF-1, we are grafted to the surface of PS materials, to examine insulin type The effect of photolytic activity growth factor I GF-1 grafting, the principle being combined using antigen-antibody, selection carries Andy FlouTM 488 The IGF-1 antibody of fluorescence carries out immunofluorescence experiment, to characterize the grafting result of support.Preferably experimental result is：Green is glimmering Light is by the center of presentation at intervals of 25 μm of light and dark striped.As shown in figure 5, we arrive in timbering material surface observation really Corresponding experimental phenomena：Zonal distribution is presented in green fluorescence；Strip width size is homogeneous, about 12 μm；Heart septum is equal in band It is even, about 25 μm.As shown in figure 5, we use λ=250nm, D=79cm, d=1cm, a=5mm, b=5mm to be carried out for condition Experiment.
After synthesis photolytic activity growth factor I GF-1, we are grafted to the surface of PS materials, and utilize antigen-antibody The principle being combined, selection carries Andy FlouTMThe IGF-1 antibody of 488 fluorescence carries out immunofluorescence experiment, to characterize support Grafting result.Preferable experimental result should be：IGF-1 is grafted on material into " ten " shape striped, regionality.Such as Fig. 5 institutes Show, we have arrived corresponding experimental phenomena in timbering material surface observation really：" ten " shape fringe distribution is presented in green fluorescence.
The protein expression of the Immunofluorescence test cell of embodiment 5
1st, cell culture
Bare stent and 1, the support through modification are respectively put into 24 porocyte culture plates, cell is merged in blake bottle Cultivate to after 60~90%, with 1~2 × 104The density in/hole is seeded on 24 orifice plates, culture 3,6,9 days, to carry out follow-up reality Test.Other cell culture conditions are：Low sugar DMEM culture mediums containing 10% NBCS, 37 DEG C, 5.0%CO2。
2nd, the immune detection of cell differentiation
After cell culture in 24 orifice plates, PBS solution shaking table is cleaned 3~5 times, every time after 5~10min, using 2~5% Paraformaldehyde is fixed after 30~40min, PBS, and experimental group is divided into two：Directly dyed or vitellophag after be made Cell smear (smear methods：After 4~7min of pancreatin immersion sample, serum-containing medium terminates digestion, and dropper blows and beats support, Cell is collected by centrifugation in 2500~3000rpm/min, is coated in load fragment).20~30min of 0.2%Triton X-100 permeabilizations； After PBS is cleaned again, lucifuge is incubated fluorescence antibody 2~3h, DAPI dye liquor and is incubated 3~5min.PBS, microscopy.
3rd, result
(1) immunofluorescence results of cell differentiation are as shown in Figure 6.
Experimental result in Fig. 5, this kinds of IGF-1 is gone up when being grafted on PS supports in the method for the ultraviolet interference of light and diffraction When insulin-like growth factor, the alternate shape distribution of striped and the distribution of " ten " shape can be presented in this kind of growth factor of IGF-1 (see Fig. 5).Mesenchymal stem cells MSCs is grafted on the support, while with SD rat bone marrow mesenchymal stem cellses osteogenic inductions Differential medium goes culture 3 weeks.And the principle being combined using antigen-antibody, carries out immunofluorescence real to the cell on support Test.It was found that, the differentiation of mesenchymal stem cells MSCs can distribution along this kind of factor of IGF-1 on support, it is zonal into Osteocyte direction breaks up.Can the as can be seen clearly from figure 6 characteristic protein BGP albumen of Gegenbaur's cell distribution be according to Distribution of this kind of factor of IGF-1 on support.Also just explanation compartmentalization photo-grafting support has to mesenchymal stem cells MSCs The different parts for inducing the cell of different zones on the effect of differentiation, support to be combined with growth factor, and carry out different portions The differentiation of position, to reach the purpose of compartmentalization differentiation.
(2) immunoblot results of cell differentiation are as shown in Figure 7.
Experimental result in Fig. 4 and Fig. 5, IGF- is gone up when being grafted on PS supports in the method for the ultraviolet interference of light and diffraction When 1 this kind of factor, the cellular portions on support can break up to Gegenbaur's cell direction, and also part cell maintains BMSC's Physiological status.The BMSC of Gegenbaur's cell can be divided into as apparent from Fig 7 the synthesis of part BGP.Compared to control group For, control group substantially only has the expression of a small amount of BGP, i.e. cell not to break up completely to Gegenbaur's cell direction, and singly stitches and spread out Penetrate with two groups of the two-slit interference expression for having 31.99% and 30.55% respectively for internal reference β-actin, show BMSC Truly have partial differentiation for Gegenbaur's cell.From the point of view of Fig. 6, the region that BMSC has been grafted growth factor in region is differentiated , BMSC characteristic is still remain without being grafted the region of growth factor.

Claims (10)

1. a kind of method using the ultraviolet interference of light/diffraction characteristic area light graft growth factor, it is characterised in that be to occur In the presence of the ultraviolet light of interference or diffraction, the regionality of insulin-like growth factor 1 is grafted to bone tissue engineer two-dimensional material Surface, structure obtains regional photo-grafting support.
2. according to the method described in claim 1, it is characterised in that the bone tissue engineer two-dimensional material is high molecular polymer Class two-dimensional material.
3. according to the method described in claim 1, it is characterised in that the bone tissue engineer two-dimensional material be polystyrene, PCL-PLA or PLGA.
4. according to the method described in claim 1, it is characterised in that the bone tissue engineer two-dimensional material is 1~3cm's of diameter Polystyrene two-dimensional material.
5. according to the method described in claim 1, it is characterised in that the insulin-like growth factor 1 is first passed through after photoactivation Photo-grafting to two-dimensional material surface, the condition of photo-grafting be interfere or 220~260W uviol lamps of diffraction under handle two Secondary, processing time is 10~30min.
6. according to the method described in claim 1, it is characterised in that comprise the following steps：
S1. photolytic activity IGF-1 is prepared：Under the conditions of lucifuge, IGF-1 is added to the DMF/PBS solution of the succinimide ester containing N- In, 40~50h of stirring reaction under conditions of 0~4 DEG C；Centrifugal purification；
S2. it is modified bone tissue engineer two-dimensional material
Spontaneously dried under the conditions of photolytic activity IGF-1 solution, lucifuge is added dropwise to bone tissue engineer two-dimensional material surface, after being dried Support；Dry after-poppet to be placed in optical system, 10~30min is handled under 220~260W uviol lamps；Deionized water rinsing 1~ 6min, natural air drying；Natural drying under the conditions of photolytic activity IGF-1 solution lucifuges is added dropwise again, after-poppet conversion angle is dried, then It is secondary to be placed in the optical system, 10~30min is handled under 220~260W uviol lamps, regional photo-grafting support is obtained；Its In, the optical system can realize two-slit interference or the single slit diffraction of ultraviolet light.
7. method according to claim 6, it is characterised in that in step S1, IGF-1：The succinimide ester containing N- The mass volume ratio of DMF/PBS solution is 40~60 μ g：10~30ml.
8. method according to claim 6, it is characterised in that bone tissue engineer two-dimensional material described in step S2 is diameter 1 ~3cm circular PS materials.
9. the regional photo-grafting support prepared according to any methods described of claim 1~8.
10. regional photo-grafting support described in claim 9 as or application in terms of preparing bone tissue engineering scaffold.
CN201710335943.3A 2017-05-12 2017-05-12 A kind of application of method and its bone tissue engineering scaffold modification of the area light graft growth factor Pending CN107261204A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101193666A (en) * 2005-04-26 2008-06-04 奥胡斯大学 Biocompatible material for surgical implants and cell guiding tissue culture surfaces
CN103908703A (en) * 2014-04-28 2014-07-09 华南师范大学 Anti-cell senescence bone tissue engineering support material and application thereof
CN104826162A (en) * 2015-05-26 2015-08-12 华南师范大学 Preparation and application of PCL-PLA (polycaprolactone-polylactic acid) tissue engineering composite scaffold with liver cell anti-aging function
US9480773B2 (en) * 1999-03-19 2016-11-01 The Regents Of The University Of Michigan Mineralization and biological modification of biomaterial surfaces

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9480773B2 (en) * 1999-03-19 2016-11-01 The Regents Of The University Of Michigan Mineralization and biological modification of biomaterial surfaces
CN101193666A (en) * 2005-04-26 2008-06-04 奥胡斯大学 Biocompatible material for surgical implants and cell guiding tissue culture surfaces
CN103908703A (en) * 2014-04-28 2014-07-09 华南师范大学 Anti-cell senescence bone tissue engineering support material and application thereof
CN104826162A (en) * 2015-05-26 2015-08-12 华南师范大学 Preparation and application of PCL-PLA (polycaprolactone-polylactic acid) tissue engineering composite scaffold with liver cell anti-aging function

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