CN108398381A - The method of non-invasive dynamic monitoring material degradation rate and regeneration matching degree - Google Patents
The method of non-invasive dynamic monitoring material degradation rate and regeneration matching degree Download PDFInfo
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Abstract
A kind of method of degradation rate and regeneration matching degree in non-invasive dynamic monitoring material bodies carries out Double-mode imaging to the material in implantation biological subject body simultaneously using one-dimensional image method and three-D imaging method.Monitoring method provided by the invention can not only be monitored from one-dimensional plane, can be monitored from three-D space structure, can be established monitoring timbering material degradation rate and be organized the formation of the system of matching degree, realize the non-invasive and dynamic of monitoring.
Description
Technical field
The present invention relates to a kind of imaging that implantation material is degraded in vivo and monitoring method more particularly to a kind of monitorings
Material degradation rate and the method for organizing the formation of matching degree realize the dynamic of monitoring and non-invasive.
Background technology
Organizational project basic principle is that the seed cell of amplification in vitro is inoculated into biodegradable bracket material, passes through cell
The gradually degradation of proliferation and Extracellular Matrix Secretion and timbering material on timbering material, to final shape in vitro or in vivo
At engineered tissue.In this course, timbering material provides not only cell adherence, migration, proliferation and functional metabolism,
Also carry cell and signaling molecule and the functions such as the transmission of nutriment and the excretion of waste are unobstructed.
One of the important feature of ideal stent material is that have and organize the formation of the degradation rate to match.Timbering material mistake
Early degradation will be unable to provide required holder by cytothesis tissue；It degrades too late, the effect that causes to occupy makes cell without space
Regenerating tissues.However in practical study, how whether dynamic monitoring timbering material degradation rate matches still with organizing the formation of
It is a this field one of problem urgently to be resolved hurrily.
Traditional research method is that degradation situation and the regeneration of timbering material are determined using tissue section strain method
Situation.But this method is depended on puts to death Different Individual animal in set time node, cannot achieve the company in same animal body
Continuous observation.In recent years, non-intruding imaging technique (Non-invasive Imaging Technology, NIR) is sent out rapidly
Exhibition, including X ray computer fault imaging (CT), ultrasonic imaging (USI), magnetic resonance imaging (MRI) and fluorescent optics imaging
Technology etc. provides feasibility for the degradation of dynamic monitoring in-vivo tissue engineering scaffold material.However, large biological molecule or macromolecule
The tissue engineering bracket materials such as polymer can not use the above method to be imaged or develop.
Chinese invention patent ZL200910026721.9 discloses a kind of preparation of biodegradable fluorescent polyester multipolymer
Method, using 3,4-dihydroxycinnamic acid as main monomer, polyethylene glycol PEG400, PEG1000 of different molecular weight, PEG2000,
PEG4000, PEG6000, lactic acid, P-hydroxybenzoic acid or lithocholic acid are function monomer, and sodium acetate is catalyst, and acetic anhydride is
Solvent has obtained caffeic acid ester analog copolymer using two step melt-polycondensations.Prepared caffeic acid ester analog copolymer can be used as
Degradable material or fluorescence probe are applied to the fields such as organizational project, biological medicine and environmental protection.
It is poly- that Chinese invention patent application 201410711669.1 discloses a kind of fluorescent degradable block of grafting of tool
Urethane, bone renovating material and preparation method, in the hard section by aliphatic diisocyanate, with the polymer containing terminal hydroxy group or embedding
It is grafted with acceptable fluorescence in medicine in the structure that the soft segment of the degradable polymer segment of section copolymer is polymerized
Ingredient.The bone renovating material polyurethane is collectively constituted with nanometer hydroxyapatite powder, has good biocompatibility and can
Degradability, and there is fluorescent characteristic simultaneously, the degradation process of tracer evaluation block polyurethane is can be used to, mechanism of degradation is analyzed, examines
Influence of the degradation rate to material mechanical performance and regeneration reconstruction process is examined, is the biological safety of macromolecule degradable material
Evaluation provides new visual angle and means.
2013, Soon Hee Kim of Harvard University etc. just for the first time report utilize mouse model, by both sexes from
Sub- near-infrared fluorescent nano molecular probe ZW800-1 mark collagen scaffold and it has been carried out for a long time in vivo near-infrared fluorescent at
Picture.The result shows that fluorescence imaging can the dynamic process (Scientific that is metabolized in vivo of tracer collagen scaffold in real time
reports 2013；3).
With the degradation of timbering material, the material monomer and fluorescent nano probe of disintegration will be released and remove, and repair area
The fluorescence in domain can be more and more weaker, until holder is degradable and disappears.And in timbering material degradation process, cambium regeneration
The density and volume of (such as at cartilage or skeletonization) are increasing, until repairing entire impingement.In this course, holder material
Among the free fluorescent marker discharged after material degradation can enter cambium by peripheral cell phagocytosis, cambium is made to have
There is fluorescent marker, so as to cause the fluorescence signal on undegradable timbering material cannot be distinguished in fluoroscopic imaging systems.
Therefore, although fluorescent molecular probe and fluoroscopic imaging systems can be to the degradation situations of timbering material in cell and molecule
Tracer is carried out in level, and there is very high sensitivity, but it is disadvantageous in that and can only be imaged on one-dimensional plane, it can not
Acquisition live body three-dimensional structure (section view) and three dimensions resolution ratio are low etc..Meanwhile fluorescence probe is possible to quilt in metabolism
Peripheral cell absorbs, and hence into cambium, when fluorescence is remained wherein, how to distinguish the residual fluorescence and original of cambium
Timbering material fluorescence becomes and the insurmountable problem of fluorescence imaging is used alone.
Invention content
It is an object of the present invention to provide it is a kind of monitoring material degradation rate method, realize monitoring dynamic and
It is non-invasive.
It is monitored it is another object of the present invention to provide the situation by organizational project formed tissue in vivo
Method, realize the dynamic of monitoring and non-invasive.
It is yet a further object of the present invention to provide a kind of sides for monitoring material degradation rate and organizing the formation of matching degree
Method realizes the dynamic of monitoring and non-invasive.
A further object of the present invention is to provide noninvasive in Bone Defect Repari and dynamic monitoring material degradation rate, tissue shape
At and its matching degree method.
A kind of monitoring material degradation rate provided by the invention and the method for organizing the formation of matching degree, using one-dimensional image
Method and three-D imaging method carry out Double-mode imaging to the material in implantation biological subject body simultaneously.One-dimensional image method is such as：
But it is not limited only to fluorescence imaging method and isotope imaging method.Three-D imaging method is such as：But it is not limited only to CT imaging methods, magnetic
Resonant imaging method, PET imaging methods and B ultrasound imaging method etc..
Monitoring method provided by the invention can be used for tissue repair (such as：Ostosis), to monitor the degradation of timbering material
Rate, the state and its matching degree of regeneration.
One-dimensional image method is (such as：Fluoroscopic imaging systems) and three-D imaging method is (such as：Micro CT) composition double mode
Imaging not only can be from one-dimensional plane (such as：Fluorescence) monitoring, while can be from three-D space structure (such as：Micro CT micro-imagings)
It is monitored, the use that the two combines has complementary effect.
Be implanted into biological subject body in material be biodegradable material such as：But it is total to be not limited only to poly lactic-co-glycolic acid
(poly (lactic-co-glycolic acid, PLGA), marks or is mixed into the marker of (doping), such as to polymers thereon：But no
It is only limitted to fluorescent marker, magnetic particulate labels and isotopic label etc., especially fluorescence nano marker, magnetic particle nanometer
Marker and isotope Nanoparticle labeling object etc..Marker preferentially selects near-infrared fluorescent material, such as：But it is not limited only to gold nano group
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 captures ray signal from the biological subject body of implantation material.Such as：Fluorescence imaging device will be infrared
The biological subject body of light (especially near infrared light) irradiation implantation material, the optical signal after being irradiated by captured by camera.
Three-dimensional imaging device captures ray signal from the biological subject body of implantation material.Such as：Micro- CT equipment is by roentgenogram
The biological subject body for penetrating implantation material obtains monitoring signals by X-ray detector and is imaged.
The advantageous effect that technical solution of the present invention is realized：
Method provided by the invention combines both one-dimensional image method and three-D imaging method, while tested to being implanted into
Material in organism carries out Double-mode imaging, can not only be monitored from one-dimensional plane, while can be supervised from three-D space structure
It surveys, monitoring timbering material degradation rate can be established and organizes the formation of the system of matching degree, realize the non-invasive and dynamic of monitoring
State property.
Method provided by the invention can also solve the differentiation of fluorescence under specific condition, i.e., to free and on timbering material
Fluorescence signal realize and distinguish, solve the interference problem that is fluoresced to former timbering material in cambium, reach effective differentiation
The purpose of timbering material and cambium, and finally solve the degradation rate of timbering material and regeneration matches the difficulty of monitoring
Topic.
Description of the drawings
Fig. 1 is the schematic diagram of one embodiment of method of present invention monitoring material degradation rate；
Fig. 2 is CT figures when nude mice by subcutaneous is implanted into degradation material；
Fig. 3 is the degradation CT figures that degradation material is implanted into after nude mice by subcutaneous；
CT when Fig. 4 is damaged for Rat calvarial schemes, and arrow show impaired place in figure；
Fig. 5 is to scheme in the CT of the impaired place's implantation degradation material in situ tissue reparation of Rat calvarial, and arrow is shown in figure
State after impaired place is repaired.
Specific implementation mode
Below in conjunction with attached drawing detailed description of the present invention technical solution.The embodiment of the present invention is only to illustrate the skill of the present invention
Art scheme and it is unrestricted, although being described the invention in detail with reference to preferred embodiment, those skilled in the art
It should be appreciated that can be modified or replaced equivalently to the technical solution of invention, without departing from the essence of technical solution of the present invention
God and range, should all cover in scope of the presently claimed invention.
It has been investigated that Micro CT imaging can only to sclerous tissues, such as：The tissues such as bone and tooth have good imaging
Effect, and to low density soft tissue such as liver, heart, blood vessel and gastrointestinal tract etc. and the imagings such as high molecular material or polymer
Effect is poor, cannot be distinguished between tissue, and some cannot be imaged.It is template using bovine serum albumin (BSA) or egg white etc., at
Work(it is prepared for a series of noble metal fluorescence nano clusters (Au, Ag, Pt Cluster) with good biocompatibility
(Nanoscale Research Letters, 2013,8：182；Nanoscale 2012,4 (24)：7766), experimental result table
Bright, gold nanoparticle or nanocluster all have good CT imaging effects in toy angiography and tumor imaging
(Optics Express 2011,19 (18)：17030；J.Mater.Chem.B, 2014,2 (40)：6931-6938；
J.Mater.Chem.B, 2013,1 (38)：5045-5053).Gold nanoparticle is effective soft tissue CT contrast agent, and Jenner
Rice cluster has good fluorescent characteristic, complies fully with the requirement of fluorescence and Micro CT Double-mode imagings.
Currently, in materials science field, the surface modifications such as inorganic nano material or high-molecular organic material or modification, one
As with direct absorption, Electrostatic Absorption, coupling agent covalent linkage etc..Special high molecular polymer, such as：The surface hydrophilic of PLGA
Sex modification.Alkaline matter is added in PLGA and forms alkaline solution, the hydroxide ion in solution and polymer material molecule bone
Ester bond in frame reacts, and causes the part ester bond in polymer molecular chain to be broken, to make surface of polymer material
Hydrophilic radical-the carboxyl and hydroxyl of exposure are formed, the appearance of these hydrophilic radicals and increasing helps to improve the hydrophily of PLGA,
The cell adhesion of reinforcing material.Therefore, the fluorescence Jenner with aminoterminal is covalently attached by coupling agent on the carboxyl of PLGA
Rice cluster.Chemical coupling method has many advantages, such as to be firmly combined with, quantitative relationship is apparent.
It is specific as follows that chemical method produces the step of fluorescent marker PLGA materials：PLGA timbering material 0.1g are first taken, are added
The sodium hydroxide solution 50mL of 10mM forms alkaline solution, in the hydroxide ion and polymer material molecular skeleton in solution
Ester bond react, cause ester bond in polymer molecular chain that portion fractures occur, to make PLGA material surfaces be formed cruelly
Hydrophilic radical-the carboxyl and hydroxyl of dew, provide the binding site of marking nano cluster.
Mixing in 500 μ L connection buffer solutions is added in the nanocluster of the NHS of EDC, 0.1mg of 2mg and 0.2mg, is kept away
Light continuous rotation is reacted 30 minutes, and the PLGA materials that the carboxylated prepared is added after the amino of nanocluster, the company of being protected from light are activated
Continuous revolving reaction 24 hours.Solution is collected with 5 × 103Rpm centrifuge washings twice, 15 minutes every time, discard supernatant liquid to remove
Excessive unreacted nanocluster in solution finally has near-infrared fluorescent gold nano cluster PLGA materials cold by what is prepared
It is lyophilized dry with spare.
Directly doping and 3 D-printing method also can be used and prepare fluorescence PLGA.Detailed process is as follows：(1) by different molecular weight
PLGA melt at high temperature, then take various concentration fluorescence gold nano cluster be added melt PLGA solution in, be sufficiently stirred,
With spare；(2) animal pattern repairs the acquisition of area to be repaired micro-data：Take Rat calvarial sample, remove surface skin and
Soft tissue.(μ CT-80) is scanned using Micro-CT, scanning threshold value is set as 220Hu.Unenhanced data reach graphic processing data work
It stands, preserves all data in dicom format；(3) according to DICOM data screenings and stl file is generated：By the number of Micro-CT
According to medical threedimensional images processing software Mimics is imported, bone, cartilage, soft tissue are offered a clear explanation according to gray scale and isolate bone
The 3-dimensional image data of bone can see the bionical skull defeci mould of height that numerical data is built after determining resolution ratio and precision
Type；(4) skull rapid shaping：Stl file is the three-dimensional data format that can be identified at present by most of RP (rapid shaping) machine,
The stl file of Mimics software editings is sent to rapidform machine (offer of Hangzhou 3D-Print companies), is finally received with fluorescence
The cluster doped PLGA of rice is by its printing shaping.
Fig. 1 is the present invention with one-dimensional image method and three-D imaging method while to the material in implantation biological subject body
Carry out an embodiment schematic diagram of the Double-mode imaging to monitor material degradation rate Yu organize the formation of matching degree.Fluorescence obtained
Label or the PLGA materials of doping are implanted into biological subject body, and fluorescence imaging device is applied simultaneously in specific time and time interval
Biological subject body is monitored with micro- CT equipment (i.e. Double-mode imaging technology), understands the degradation rate and group of implantation material
It knits to form situation and its matching degree.
Fluorescence imaging device is that near infrared imaging system is completed in improved Kodak living imaging instruments.
AndorDU897EMCCD is as signal receiver.Near-infrared fluorescent image is analyzed by Kodak molecular imaging softwares.Swash
For light emitting source using the continuous wave near infrared laser in addition introduced, emission spectrum capture range is 600nm-1100nm.Tested life
Object is (such as：Nude mice) using progress live body near infrared imaging observation after 10% chloral hydrate anesthesia.
Micro CT imaging uses the MicroCT μ CT80 types of SCANCO Medical AG companies of Switzerland.It is solid after nude mice anesthesia
It due in sample cell, using 20 μm of resolution ratio, is quickly scanned, last three-dimensional reconstruction.Analyze experimental group and control group
The degradation situation of PLGA nude mice by subcutaneous.
1 nude mice by subcutaneous of embodiment is implanted into (dystopy) degradation model dynamic monitoring
Nude mice 70 is chosen, is divided into 4 groups, respectively at the identical above-mentioned fluorescence PLGA of nude mice by subcutaneous plantation volume as experiment
The PLGA of group and non-marked is as a control group.Wherein two groups for Double-mode imaging technology continuously dynamically observation (experimental group 5+
Control group 5=10), observation is primary every two weeks, observes 3 months, totally 6 Double-mode imaging monitorings, gathered data.Other two
Group corresponds to dynamic for tissue section strain method verification double mode monitoring result (30+control group of experimental group 30=60 is only)
The time of monitoring, sampling is primary every two weeks, takes experimental group and each 5 of control group every time, does tissue section strain detection support material
The degradation situation of material analyzes data.
According to the timing node of selection, collect respectively corresponding near infrared imaging and Micro CT imaging result (referring to Fig. 2 and
Fig. 3), it is analyzed through image software, the variation according to imaging signal calculates the skeletonization of corresponding degradation rate and cambium
Effect.
The foundation (ira situ degradation model) and its dynamic monitoring of 2 rat tissue's engineering skull regeneration model of embodiment
Kunming rat 70 is chosen, is divided into 4 groups, a diameter of 5mm skull defecis are drilled through on Rat calvarial after anaesthetizing respectively
(referring to Fig. 4) takes above-mentioned fluorescence PLGA to be tested as a control group as the PLGA of experimental group and non-marked, respectively will experiment
Group and control group timbering material are filled into skull defeci, are then sewed up a wound.Wherein two groups are used for Double-mode imaging technical battery
Continuous dynamic observation (5+control group of experimental group 5=10), observation is primary every two weeks, observes 3 months, totally 6 double modes at
As monitoring, gathered data.Other two groups are verified double mode monitoring result (experimental group 30+control for tissue section strain method
Group 30=60 is only), correspond to the time of dynamic monitoring, sampling is primary every two weeks, takes experimental group and each 5 of control group every time, does
The degradation situation of tissue section strain detection support material analyzes data.
The near-infrared spectroscopy at transfer operation position and the variation feelings of Micro CT imaging in observation animal model body in real time
Condition determines degradation rate and the regenerated process of cambium and feature in fluorescence PLGA timbering material bodies.According to the time of selection
Node collects corresponding near infrared imaging and Micro CT imaging as a result, analyzing to come through image software, according to imaging signal respectively
Variation calculate the skeletonization effect of corresponding degradation rate and cambium (referring to Fig. 5).
Claims (10)
1. a kind of method of monitoring, it is characterised in that using one-dimensional image method and three-D imaging method simultaneously to being implanted into tested life
Material in object carries out Double-mode imaging.
2. monitoring method according to claim 1, it is characterised in that the one-dimensional image method is selected from fluorescence imaging
The one or more of method and isotope imaging method.
3. monitoring method according to claim 1, it is characterised in that the three-D imaging method is selected from the imaging sides CT
The one or more of method, MR imaging method, PET imaging methods and B ultrasound imaging method.
4. monitoring method according to claim 1 is used to monitor the degradation rate of timbering material.
5. monitoring method according to claim 1 is used for the regenerated state of monitoring tissue.
6. monitoring method according to claim 1 is for monitoring material degradation rate and regeneration matching degree.
7. application of the monitoring method according to claim 1 in tissue repair.
8. monitoring method according to claim 1, it is characterised in that the material is the life of label or doping marker
Biodegradable material.
9. monitoring method according to claim 1, it is characterised in that the marker is selected from fluorescent marker, magnetic
The one or more of grain marker and isotopic label.
10. monitoring method according to claim 1, it is characterised in that the marker is marked selected from fluorescence nano
The one or more of object, magnetic particle Nanoparticle labeling object and isotope Nanoparticle labeling object.
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