CN104880408A - Method for non-invasively and dynamically monitoring matching degree of material degradation rate and tissue regeneration - Google Patents
Method for non-invasively and dynamically monitoring matching degree of material degradation rate and tissue regeneration Download PDFInfo
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Abstract
The invention provides a method for non-invasively and dynamically monitoring the matching degree of material degradation rate and tissue regeneration. According to a one-dimensional imaging method and a three-dimensional imaging method, double-mode type imaging is carried out on materials implanted into a tested living body. The monitoring method provided by the invention not only can be used for monitoring from a one-dimensional plane, but also can be used for monitoring from a three-dimensional plane; and a system for monitoring the matching degree of material degradation rate and tissue regeneration can be established, and the non-invasiveness and the dynamic property of monitoring are realized.
Description
Technical field
The present invention relates to imaging and monitoring method that a kind of embedded material degrades in vivo, particularly relate to a kind of method of monitoring material degradation speed and organization formation matching degree, realize the dynamic of monitoring and non-invasive.
Background technology
Organizational project ultimate principle is inoculated in biodegradable bracket material by the seed cell of amplification in vitro, by the propagation of cell on timbering material and the progressively degraded of Extracellular Matrix Secretion and timbering material, thus final formative tissue engineering tissue in vitro or in body.In this course, timbering material provide not only cell adherence, migration, proliferation and function metabolism, also carry the functions such as the excretion of the transmission of cell and signaling molecule and nutriment and refuse is unobstructed.
One of key character of ideal stent material has the degradation rate matched with organization formation.Timbering material premature breakdown cannot provide required support to organize by cytothesis; Crossing degraded in the evening effect that then causes occupying makes cell organize without spacing regenerative.But in practical study, how whether dynamic monitoring timbering material degradation rate and organization formation mate and remain one of this area difficult problem urgently to be resolved hurrily.
Traditional research method adopts tissue section strain method to determine degraded situation and the regeneration situation of timbering material.But this method depends on stubbornly refuses same individual animals at set time Nodes, cannot realize the Continuous Observation in same animal body.In recent years, non-intruding imaging technique (Non-invasive Imaging Technology, NIR) developed rapidly, comprise X ray computer fault imaging (CT), ultrasonic imaging (USI), magnetic resonance imaging (MRI) and fluorescent optics imaging technique etc., for the degraded of dynamic monitoring in-vivo tissue engineering scaffold material provides feasibility.But the tissue engineering bracket material such as biomacromolecule or high molecular polymer cannot adopt said method imaging or development.
Chinese invention patent ZL200910026721.9 discloses a kind of preparation method of biodegradable fluorescent polyester multipolymer, with 3,4-dihydroxycinnamic acid is main monomer, polyglycol PEG400, the PEG1000 of different molecular weight, PEG2000, PEG4000, PEG6000, lactic acid, P-hydroxybenzoic acid or lithocholic acid are function monomer, sodium acetate is catalyzer, acetic anhydride is solvent, adopts two step melt-polycondensations to obtain caffeic acid ester analog copolymer.Prepared caffeic acid ester analog copolymer can be used as degradable material or fluorescence probe is applied to the fields such as organizational project, biological medicine and environmental protection.
Chinese invention patent application 201410711669.1 discloses one and has epipolic grafting degradable block polyurethane, bone renovating material and preparation method, in the hard section by aliphatic diisocyanate, be grafted with acceptable fluorescence composition in medical science with the soft section of structure be polymerized of the degradable polymer segment of the polymkeric substance containing terminal hydroxy group or segmented copolymer.This polyurethane of bone renovating material and nanometer hydroxyapatite powder form jointly, there is good biocompatibility and degradability, and there is fluorescent characteristic simultaneously, can be used to the degradation process that block polyurethane is evaluated in spike, analyze mechanism of degradation, investigate the impact of degradation rate on material mechanical performance and regeneration process of reconstruction, the Evaluation of Biocompatibility for macromolecule degradable material provides new visual angle and means.
2013, the Soon Hee Kim of Harvard University etc. just reported first utilized mouse model, mark collagen scaffold and carried out near-infrared fluorescence imaging in long-time body to it by zwitter-ion near-infrared fluorescent nano molecular probe ZW800-1.Result shows, fluorescence imaging can dynamic process (the Scientific reports 2013 of the collagen scaffold of spike in real time metabolism in vivo; 3).
Along with the degraded of timbering material, the material monomer of disintegration and fluorescent nano probe will be released and remove, and the fluorescence of restoring area can be more and more weak, until support is degradable and disappear.And in timbering material degradation process, density and the volume of cambium regeneration (as become cartilage or skeletonization etc.) are increasing, until repair whole impingement.In this course, the free fluorescent marker discharged after timbering material degraded can be engulfed by peripheral cell and enter among cambium, make cambium have fluorescence labeling, thus cause the fluorescence signal that cannot distinguish in fluoroscopic imaging systems on undegradable timbering material.
Therefore, although fluorescent molecular probe and fluoroscopic imaging systems can carry out spike to the degraded situation of timbering material on cell and molecular level, there is very high sensitivity, can only imaging on one-dimensional plane but its weak point is, live body three-dimensional structure (analysing and observe property) cannot be obtained and three dimensions resolution low inferior.Meanwhile, fluorescence probe is likely absorbed by peripheral cell when metabolism, thus enters in cambium, when fluorescence remains wherein, how to distinguish the residual fluorescence of cambium and former timbering material fluorescence, becomes and be used alone the insurmountable difficult problem of fluorescence imaging.
Summary of the invention
One object of the present invention is to provide a kind of method of monitoring material degradation speed, realizes the dynamic and non-invasive of monitoring.
Another object of the present invention is to provide carries out the method for monitoring by the situation of organizational project institute's formative tissue in vivo, realizes the dynamic of monitoring and non-invasive.
Another object of the present invention is to provide a kind of method of monitoring material degradation speed and organization formation matching degree, realizes the dynamic and non-invasive of monitoring.
Another object of the present invention is to be provided in the method without wound and dynamic monitoring material degradation speed, organization formation and matching degree thereof in Bone Defect Repari.
A kind of method of monitoring material degradation speed and organization formation matching degree provided by the invention, adopts one-dimensional image method and three-D imaging method to carry out Double-mode imaging to the material implanted in biological subject body simultaneously.One-dimensional image method as: but be not limited only to fluorescence imaging method and isotope formation method.Three-D imaging method as: but be not limited only to CT formation method, MR imaging method, PET formation method and B ultrasonic formation method etc.
Monitoring method provided by the invention can be used for organization restoration (as: ostosis), to monitor the degradation rate of timbering material, the state of regeneration and matching degree thereof.
The Double-mode imaging that one-dimensional image method (as: fluoroscopic imaging systems) and three-D imaging method (as: Micro CT) form not only can be monitored from one-dimensional plane (as: fluorescence), can monitor from three-D space structure (as: Micro CT micro-imaging), the two use combined has complementary effect simultaneously.
The material implanted in biological subject body be Biodegradable material as: but be not limited only to Poly(D,L-lactide-co-glycolide (poly (lactic-co-glycolic acid, PLGA), it marks or is mixed into the label of (doping), as: but be not limited only to fluorescent marker, magnetic particulate labels and isotopic label etc., especially fluorescence nano label, magnetic particle Nanoparticle labeling thing and isotope Nanoparticle labeling thing etc.Label prioritizing selection near-infrared fluorescent material, as: but be not limited only to gold nano cluster (Nanoscale 2012,4 (24): 7766-72; Nano Res.2012,5 (9): 630-639; Nanoscale Res.Lett.2013,8 (1): 182).
One-dimensional image equipment catches ray signal from the biological subject body of embedded material.As: infrared light (especially near infrared light) is irradiated the biological subject body of embedded material by fluorescence imaging device, by the postradiation light signal of captured by camera.
Three-dimensional imaging device catches ray signal from the biological subject body of embedded material.As: Micro-CT scanning equipment, by the biological subject body of x-ray bombardment embedded material, obtains monitor signal and imaging by X-ray detector.
The beneficial effect that technical solution of the present invention realizes:
Method provided by the invention, both one-dimensional image method and three-D imaging method are combined, Double-mode imaging is carried out to the material implanted in biological subject body simultaneously, not only can monitor from one-dimensional plane, can monitor from three-D space structure simultaneously, the system of monitoring timbering material degradation rate and organization formation matching degree can be set up, realize the non-invasive and dynamic of monitoring.
Method provided by the invention can also solve the differentiation of fluorescence under specific condition, namely realize distinguishing to free and on timbering material fluorescence signal, solve in cambium the fluorescigenic interference problem of former timbering material, reach the object effectively distinguishing timbering material and cambium, a difficult problem for the monitoring and the degradation rate of final solution timbering material and regeneration match.
Accompanying drawing explanation
Fig. 1 is the schematic diagram that the present invention monitors method one embodiment of material degradation speed;
CT figure when Fig. 2 is nude mice by subcutaneous implantation degradation material;
Fig. 3 is that the degraded CT after degradation material implants nude mice by subcutaneous schemes;
Fig. 4 be Rat calvarial impaired time CT figure, in figure, arrow is depicted as impaired place;
Fig. 5 is the CT figure implanting degradation material original position organization restoration at the impaired place of Rat calvarial, and in figure, arrow is depicted as the state of impaired place after repairing.
Embodiment
Technical scheme of the present invention is described in detail below in conjunction with accompanying drawing.The embodiment of the present invention is only in order to illustrate technical scheme of the present invention and unrestricted, although with reference to preferred embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that, can modify to the technical scheme of invention or equivalent replacement, and not departing from the spirit and scope of technical solution of the present invention, it all should be encompassed in right of the present invention.
Find after deliberation, Micro CT imaging can only to sclerous tissues, such as: the tissue such as bone and tooth has good imaging effect, and to low density soft tissue as liver, heart, blood vessel and intestines and stomach etc. and the imaging effect such as macromolecular material or polymkeric substance poor, cannot distinguish between tissue, and some can not imaging.Utilize bovine serum albumin (BSA) or egg etc. for template, successfully prepare a series of noble metal fluorescence nano cluster (Au, Ag, Pt Cluster) (Nanoscale Research Letters with good biocompatibility, 2013,8:182; Nanoscale 2012,4 (24): 7766), experimental result shows, golden nanometer particle or nanocluster all have good CT imaging effect (Optics Express 2011,19 (18): 17030 in toy angiogram and tumor imaging; J.Mater.Chem.B, 2014,2 (40): 6931-6938; J.Mater.Chem.B, 2013,1 (38): 5045-5053).Golden nanometer particle is effective soft tissue CT contrast preparation, and gold nano cluster has good fluorescent characteristic, meets the requirement of fluorescence and Micro CT Double-mode imaging completely.
At present, in material science, the finishing such as inorganic nano material or high-molecular organic material or modification, general utilization directly absorption, Electrostatic Absorption, the link of coupling agent covalency etc.Special high molecular polymer, such as: the surface hydrophilic sex modification of PLGA.In PLGA, add alkaline matter form alkaline solution, ester bond in hydroxide ion in solution and polymeric material molecular skeleton reacts, the part ester bond in polymer molecular chain is caused to rupture, thus make surface of polymer material form hydrophilic radical-carboxyl and the hydroxyl of exposure, the appearance of these hydrophilic radicals and increase the water wettability contributing to improving PLGA, the cell adhesion of reinforcing material.Therefore, on the carboxyl of PLGA by the link of coupling agent covalency with N-terminal fluorogold nanocluster.Chemical coupling method has advantages such as combining firmly, quantitative relationship is obvious.
The step that chemical method produces fluorescence labeling PLGA material is specific as follows: first get PLGA timbering material 0.1g, add the sodium hydroxide solution 50mL of 10mM, form alkaline solution, ester bond in hydroxide ion in solution and polymeric material molecular skeleton reacts, the ester bond generating portion in polymer molecular chain is caused to rupture, thus make PLGA material surface form hydrophilic radical-carboxyl and the hydroxyl of exposure, provide the binding site of marking nano cluster.
The nanocluster of NHS and 0.2mg of EDC, 0.1mg of 2mg is added in 500 μ L connection damping fluids and mix, lucifuge continuous rotation reacts 30 minutes, add the carboxylated PLGA material prepared after the amino of activation nanocluster, lucifuge continuous rotation reacts 24 hours.Collect solution with 5 × 10
3rpm centrifuge washing twice, each 15 minutes, abandoning supernatant, to remove unreacted nanocluster excessive in solution, finally had the freeze drying of near-infrared fluorescent gold nano cluster PLGA material with for subsequent use by what prepare.
Direct doping and 3 D-printing legal system also can be adopted for fluorescence PLGA.Detailed process is as follows: the PLGA of different molecular weight at high temperature melts by (1), then the fluorogold nanocluster getting variable concentrations adds in the PLGA solution of thawing, fully stirs, with for subsequent use; (2) animal pattern repairs the acquisition of area to be repaired micro-data: get Rat calvarial sample, removes surface skin and soft tissue.Adopt Micro-CT scanning (μ CT-80), scanning threshold value is set to 220Hu.Unenhanced data reach graphic processing data workstation, preserve all data in dicom format; (3) stl file is generated according to DICOM data screening: by the data importing medical threedimensional images process software Mimics of Micro-CT, according to gray scale, bone, cartilage, soft tissue offered a clear explanation and isolate the 3-dimensional image data of bone, after determining resolution and precision, just can see the bionical skull defeci model of height that numerical data builds; (4) skull rapid shaping: stl file is at present can by the three-dimensional data form of most of RP (rapid shaping) machine identification, the stl file of Mimics software editing is sent to rapidform machine (Hangzhou 3D-Print company provides), the PLGA finally using fluorescence nano cluster doped is by its printing shaping.
Fig. 1 is that the present invention uses one-dimensional image method and three-D imaging method to carry out Double-mode imaging to monitor an embodiment schematic diagram of material degradation speed and organization formation matching degree to the material implanted in biological subject body simultaneously.Biological subject body implanted by obtained fluorescence labeling or the PLGA material of doping, apply fluorescence imaging device and Micro-CT scanning equipment (i.e. Double-mode imaging technology) is monitored biological subject body in specific time and the time interval simultaneously, understand the degradation rate of embedded material and organization formation situation and matching degree thereof.
Fluorescence imaging device is near infrared imaging system is complete on the Kodak living imaging instrument improved.AndorDU897EMCCD is as signal receiver.Near-infrared fluorescent image is analyzed by Kodak molecular imaging software.Excitation source uses the continuous wave near infrared laser introduced in addition, and emission spectrum capture range is 600nm-1100nm.Biological subject body (as: nude mice) carries out the observation of live body near infrared imaging after adopting the chloral hydrate anesthesia of 10%.
Micro CT imaging adopts the MicroCT μ CT80 type of SCANCO Medical AG company of Switzerland.Be fixed in sample cell after nude mice anesthesia, adopt the resolution of 20 μm, carry out rapid scanning, last three-dimensional reconstruction.The degraded situation of analysis design mothod group and control group PLGA nude mice by subcutaneous.
Embodiment 1 nude mice by subcutaneous implants the dynamic monitoring of (dystopy) degradation model
Choose nude mice 70, be divided into 4 groups, respectively at the identical above-mentioned fluorescence PLGA of nude mice by subcutaneous plantation volume as experimental group and cold PLGA as a control group.Wherein two groups are used for continuous dynamic concept survey (experimental group 5+control group 5=10) of Double-mode imaging technology, and observation in every two weeks once, observes 3 months, totally 6 Double-mode imagings monitorings, image data.Double mode monitoring result (experimental group 30+control group 30=60 only) is verified for other two groups for tissue section strain method, corresponding to the time of dynamic monitoring, sampling in every two weeks once, get experimental group and each 5 of control group at every turn, do the degraded situation of tissue section strain Bracket for Inspection material, analyze data.
According to the timing node chosen, collect corresponding near infrared imaging and Micro CT imaging result (see Fig. 2 and Fig. 3) respectively, through image software analysis, the change calculations according to imaging signal goes out the skeletonization effect of corresponding degradation rate and cambium.
The foundation (ira situ degradation model) of embodiment 2 rat tissue engineering skull regenerating model and dynamic monitoring thereof
Choose Kunming rat 70, be divided into 4 groups, on Rat calvarial, drilling through diameter after anesthesia is respectively 5mm skull defeci (see Fig. 4), get above-mentioned fluorescence PLGA to test as a control group as experimental group and cold PLGA, respectively experimental group and control group timbering material are filled in skull defeci, then sew up a wound.Wherein two groups are used for continuous dynamic concept survey (experimental group 5+control group 5=10) of Double-mode imaging technology, and observation in every two weeks once, observes 3 months, totally 6 Double-mode imagings monitorings, image data.Double mode monitoring result (experimental group 30+control group 30=60 only) is verified for other two groups for tissue section strain method, corresponding to the time of dynamic monitoring, sampling in every two weeks once, get experimental group and each 5 of control group at every turn, do the degraded situation of tissue section strain Bracket for Inspection material, analyze data.
The near-infrared spectroscopy at transfer operation position and the situation of change of Micro CT imaging in real-time monitored animal model, determine the process and character of fluorescence PLGA timbering material vivo degradation speed and cambium regeneration.According to the timing node chosen, collect corresponding near infrared imaging and Micro CT imaging result respectively, analyze out through image software, the change calculations according to imaging signal goes out the skeletonization effect (see Fig. 5) of corresponding degradation rate and cambium.
Claims (10)
1. a method for monitoring, is characterized in that adopting one-dimensional image method and three-D imaging method to carry out Double-mode imaging to the material implanted in biological subject body simultaneously.
2. monitoring method according to claim 1, is characterized in that described one-dimensional image method is selected from one or more of fluorescence imaging method and isotope formation method.
3. monitoring method according to claim 1, is characterized in that described three-D imaging method is selected from one or more of CT formation method, MR imaging method, PET formation method and B ultrasonic formation method.
4. monitoring method according to claim 1 is for monitoring the degradation rate of timbering material.
5. monitoring method according to claim 1 is for monitoring the state of regeneration.
6. monitoring method according to claim 1 is for monitoring material degradation speed and regeneration matching degree.
7. the application of monitoring method according to claim 1 in organization restoration.
8. monitoring method according to claim 1, is characterized in that described material is the Biodegradable material of mark or doping label.
9. monitoring method according to claim 1, is characterized in that described label is selected from one or more of fluorescent marker, magnetic particulate labels and isotopic label.
10. monitoring method according to claim 1, is characterized in that described label is selected from one or more of fluorescence nano label, magnetic particle Nanoparticle labeling thing and isotope Nanoparticle labeling thing.
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| WO2021135058A1 (en) * | 2019-12-31 | 2021-07-08 | 元心科技(深圳)有限公司 | Evaluation method and system for corrosion degree of absorbable stent |
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