CN104841020A - Macroscopic supermolecule-assembled 3D ordered tissue engineering scaffold and preparation method thereof - Google Patents
Macroscopic supermolecule-assembled 3D ordered tissue engineering scaffold and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a macroscopic supermolecule-assembled 3D ordered structure. The prepared ordered structure can be used in a cell induction differentiation template and 3D tissue engineering scaffold materials. The introduced construction elements comprise a plurality of biocompatilbe materials such as polydimethylsiloxane, hydrogel and chitosan. Through a microimprinting method, the above materials and magnetic nanoparticles are pressed into a precast temple so that a lot of the construction elements can be prepared, and the construction elements are subjected to surface modification with specific supermolecule function groups. Through an external magnetic field, the construction elements are accurately positioned in a 3D space and are fixed to designated positions by supermolecule interaction so that 3D ordered structure controllable preparation is realized. Through modification of the appointed construction element surfaces with specific protein recognition sites, needed growth factors are introduced by intermolecular specific identification in implementation so that the specified growth factors are directionally introduced into a 3D support material.
Description
Technical field
The present invention relates to Supramolecular Assembling and ordered 3 D structure field of material preparation.More specifically, a kind of three-dimensional order tissue engineering bracket and preparation of macroscopical Supramolecular Assembling is related to.
Background technology
The controlled synthesis of ordered 3 D structure gets the attention and further investigation at multiple fields such as photonic crystal, reinforcing material and photoelectric devices.At present, what the research work in this field paid close attention to nanoscale mostly constructs primitive, and study less to more than the tens microns primitives of constructing to mm-scale, its reason is that the ordered 3 D structure between this size field prepares relative difficulty: for tradition processing, this size too little being difficult to carries out accurate operation, for emerging micro-nano technology technology, this size then causes too greatly technique complicated, consuming time very long, does not have actual operability.But the ordered 3 D structure of ten microns to a centimetre has important research meaning for tissue engineering bracket, longer-wave photons field of crystals.Especially, most cells is all within this size range, current cell culture technology is also cultivated from traditional two dimension and has been advanced to Three-dimensional cell culture, and constructing the three dimensional scaffold structure comparable with cell size can carry more close to the timbering material of organism environment for Growth of Cells.Therefore, develop ten microns of preparation methoies to a cm size ordered 3 D structure, can suitable support be provided for Growth of Cells and significance be produced to promotion Tissue Engineering Study.Up to now, due to the difficulty in preparation, ten microns of relevant reports to a cm size ordered 3 D structure are less.Such as, three dimensional biological based on 3D printing technique print (3D bioprinting) method can by inkjet printing, micro-ly to extrude and the methods such as laser assisted printing, using the liquid mixture of culture environment needed for the cells such as cell, biocompatible materials and somatomedin as " ink ", utilize the threedimensional model that computer aided technique builds, carry out organizing or organ printing and reproduce.But the problem of the method is its limited resolution, be difficult to build meticulous timbering material; Due to the particularity of printing equipment, the method has certain restriction and requirement to equipment, cell, biomaterial and somatomedin, thus constrains its extensive use in field of tissue engineering technology.Therefore, develop a kind of have universality, gentle, biocompatible ordered 3 D structure preparation method remains a major challenge.
Supramolecular Assembling, as a kind of gentleness, biocompatible and do not rely on the method for special installation, can solve the problem preferably.Since proposition, what the research of traditional Supramolecular Assembling was conceived to molecular level or nanoscale usually constructs primitive.In order to self-assembly system is extended to micron and above yardstick from molecular level, the people such as Whitesides propose the concept of " full size self assembly ", and application interface minimization of free energy principle achieves at oil-water interfaces the assembling that millimeter constructs primitive, form oldered array, the structure that this array can obtain not relying on interface by follow-up curing operation and exist.In order to remove the dependence to interface, professor's Harada group introduces cyclodextrin/diphenyl diimide supermolecule functional group on grade gel mass surface, can realize gel construct the Supramolecular Assembling of primitive and obtain self-existent assembly under simple concussion condition by Host-guest Recognition effect; They utilize illumination or the regulation and control of competition molecule realization to assembling process.In the same year, the research and development group of applicant reports diameter 17 micrometer glass fiber by the Controllable assembly of coordination multiple tooth between metal-carboxylic acid at substrate surface, constructs orderly pattern.The Supramolecular Assembling mechanism of primitive is constructed in order to understand more than micron further, we introduce the concept of flexible spacer layer, multiple rigidity can be realized and construct primitive (PDMS, polymethyl methacrylate) Supramolecular Assembling, and confirm that its assembly mechanism is multiplephase mutual effect, thus propose a kind of design principle for macroscopical Supramolecular Assembling of non-aqueous gel system.In addition, the assembling such as DNA hybridization, electrostatic interaction motive force is used to the Supramolecular Assembling of the above gel rubber system of micron.These results show that Supramolecular Assembling has from bottom to top, the advantage of experiment condition gentleness, and have the specificity assembling that abundant biocompatible molecular recognition system may be used for constructing primitive in supramolecular chemistry.But the forming process due to macroscopical super-molecule assembling body has certain randomness, construct between primitive and there are some mismatching phenomenons, thus need to develop new method to realize rule, the orientation arrangement that macroscopic view constructs primitive, to form ordered 3 D structure.
Summary of the invention
One object of the present invention is the three-dimensional order tissue engineering bracket providing a kind of macroscopical Supramolecular Assembling.
Another object of the present invention is to provide a kind of macroscopical Supramolecular Assembling to prepare the method for three-dimensional order tissue engineering bracket.
The present invention is directed to current ten microns are prepared aspect difficulty to a cm size ordered 3 D structure, and the directed problem introducing multiple somatomedin in ordered 3 D structure, propose and developed a kind of Supramolecular Assembling method to realize ten microns of preparations to one centimetre of ordered 3 D structure, and introduce the particular growth factor at specific site.Biocompatibility for constructing three dimensional structure is constructed primitive and is obtained mainly through methods such as frozen section, micro-embossing, micro Process, and its character shape, size can be adjustable within the scope of tens microns to cm size.In the preparation process of constructing primitive, the magnetic nano-particle of water heat transfer, by doping mixing, has magnetic responsiveness construct primitive to prepare.Prepared by construct primitive finishing have the functional group of supermolecular mechanism or bio-identification effect.The structure of the ordered 3 D structure of ten microns to a centimetre accurately to be located and macroscopical Supramolecular Assembling is constructed primitive and fixed realization by constructing primitive in conjunction with external magnetic field to magnetic responsiveness; Construct in process at this three dimensional structure, required somatomedin can by identifying that the specific binding site constructing primitive surface is introduced.
Obtain size by the method such as frozen section, micro-embossing, the casting of mechanical micro Process masterplate and construct primitive at the biocompatibility of ten microns to a centimetre.In the preparation of constructing primitive, introduce magnetic nano-particle by the mode of mixing and doping and construct primitive to prepare ten microns of biocompatibility to one centimetre of magnetic response.To modify with supermolecular mechanism if the complementary functional groups of Host-guest Recognition, electrostatic, hydrogen bond, coordination and bio-identification effect are as the complementary functional groups of biotin/avidin (biotin/avidin), bacillus cereus rna enzyme/bacillus cereus rna enzyme inhibitor (barnase/barstar) on prepared primitive surface of constructing by alternately stratiform package technique, the modes such as mixing of adulterating, make above-mentioned complementation construct primitive and can be interacted by supermolecule or bio-identification effect realizes macroscopical Supramolecular Assembling.And then, construct primitive in conjunction with external magnetic field to magnetic responsiveness biocompatibility to carry out accurately location and macroscopical Supramolecular Assembling and construct to magnetic responsiveness biocompatibility the mode that primitive is fixed, progressively build ten microns of ordered 3 D structures to a cm size scope from bottom to top, and the supermolecule or bio-identification site by constructing primitive surface in the process of constructing introduces somatomedin, realize the particular growth factor controlled location in a three-dimensional structure, be finally expected for cell induction differentiation masterplate and tissue engineering bracket material; The magnetic responsiveness biocompatibility that the present invention introduces is constructed primitive and is referred to: this is constructed primitive and has good biocompatibility, and outside magnetic field is deposited in case, can be controlled by magnetic response mode by external magnetic field, carries out moving and locating.
The three-dimensional order tissue engineering bracket of macroscopical Supramolecular Assembling of the present invention is stacking shape ordered 3 D structure, described ordered 3 D structure by multiple at two-dimensional directional abreast, periodically the primitive of constructing of ordered arrangement form, there is supermolecule by the upper functional group of modifying in surface separately and interact and realize two surperficial connections in two primitives of constructing contacted with each other; The size of described support is 10
-5~ 10
-2m.
Primitive ordered arrangement of constructing in the present invention refers to be in and conplanely constructs primitive, is parallel to each other and adjacent, constructs primitive spacing the arrangement of ten microns to a millimeter.
For achieving the above object, the present invention adopts following technical proposals:
1. the preparation of magnetic nano-particle
Hydro-thermal method or coprecipitation is utilized to prepare ferriferrous oxide nano-particle.
2. the preparation of primitive micron is constructed to grade magnetic responsiveness biocompatibility
Biocompatibility for millimeter and above yardstick constructs primitive, can by traditional mechanical processing masterplate in cast the liquid biological compatibility construct the molding of primitive solution and realize mass biocompatibility construct primitive preparation, realize especially by following steps:
1) in conjunction with computer aided technique, adopt the laser-engraving technique in mechanical processing industry, sheet material is engraved the array of required hollow pattern, as the masterplate preparing biocompatibility and construct primitive.Mm-scale all carries out accuracy controlling to the shape of reticle pattern, size;
2) after cleaning-drying masterplate, be fixed in smooth glass surface, prepared magnetic nano-particle be scattered in crosslinkable biocompatibility and construct in primitive liquid, finally casting is filled in the pierced pattern of masterplate;
3) by the mode of mechanical grip, form pressing plate-masterplate-base plate sandwich structure, construct primitive by mode solidified biological compatibilitys such as heating, illumination, the biocompatibility that the demoulding obtains discrete magnetic response constructs primitive.
Frozen sections is prepared micro-meter scale magnetic responsiveness biocompatibility and is constructed primitive
1) primitive is constructed in the liquid biological compatibility choosing viscosity relatively large, the crosslinkable liquid biological compatibility being dispersed with magnetic nano-particle is constructed primitive and is closely suppressed between pressing plate and base plate by " sandwich " mode of employing, and biocompatibility constructs primitive thickness by two ends clamping plate thickness adjusted.
2) construct primitive by modes such as heating or illumination to the folded liquid biological compatibility being dispersed with magnetic nano-particle thoroughly to solidify, form the thin film of desired thickness, the demoulding obtains the thin slice of desired thickness.
3) under the microscope, thin slice being cut into length and width is all the thin slice of 1:1 at grade and length-width ratio.
4) with the coated above-mentioned thin slice of freezing embedding medium OCT, setting freezing microtome step distance, the thin slice embedded is placed on the position perpendicular to the freezing microtome edge of a knife, cut under the control of stepper motor, can obtain length is in batches grade, width and be highly micron-sized strip magnetic responsiveness biocompatibility and construct primitive.
Micro-embossing legal system constructs primitive for micro-meter scale magnetic responsiveness biocompatibility
1) with the SU8 photoresist array of Ultraviolet lithography at the required figure of silicon substrate surface preparation.
2) mixture of spin coating PDMS performed polymer and cross-linking agent thereof on SU8 photoresist array, copies above-mentioned array, obtains the hole array of opposite pattern, as micro-embossing masterplate.
3) primitive is constructed in the crosslinkable liquid biological compatibility being dispersed with magnetic nano-particle to drop on above-mentioned PDMS masterplate, and cover polyethylene terephthalate (PET) thin slice, compress.
4) PDMS-biocompatibility is constructed primitive liquid mixture-PET sandwich by the compacting of nanometer hot pressing instrument, fully to fill hole array on PDMS masterplate, remove the liquid level overflowing hole and remain between PDMS masterplate and PET simultaneously.
5) cause liquid biocompatibility by heating or illumination and construct that primitive mixture is crosslinked, solidification.Open PET, because the surface energy (~ 42mN/m) of PET is high more than the surface energy (~ 22mN/m) of PDMS masterplate, so open in PET process, the biocompatibility solidified is constructed primitive and can be adhered on PET, can obtain discrete magnetic responsiveness biocompatibility and constructs primitive by peeling off and collecting.
Constructing the functional group of primitive finishing in order to distinguish magnetic responsiveness biocompatibility, a small amount of dyestuff can be mixed to distinguish in mixing material.
For without step 4) in the situation of nanometer hot pressing instrument compacting, due to the existence of liquid level between PDMS masterplate and PET, the magnetic responsiveness biocompatibility that can only be secured to after the demoulding on thin film constructs cell array, and is difficult to obtain discrete magnetic responsiveness biocompatibility and constructs primitive.
3. magnetic responsiveness biocompatibility is constructed primitive finishing supermolecule or is had the molecule of bio-identification effect, and substrate surface modifies supermolecule
1) adopt graft reaction or free-radical polymerized mode, supermolecule or the molecule with bio-identification effect are connected on polyelectrolyte respectively; Described supermolecule comprises supermolecule donor or Supramolecular Receptors; The described molecule with bio-identification effect comprises bio-identification donor or bio-identification receptor.
2) magnetic responsiveness biocompatibility is constructed primitive to be cleaned by the mode that ethanol, deionized water are ultrasonic, the polyelectrolyte solution of preparation alternately needed for layered self-packaging (LbL) process, concentration is in 0.1-1.0mg/mL scope.
3) in order to distinguish be modified with different functional groups construct primitive, LbL constructs primitive with differentiation of dye before modifying, such as redness and green.The viridescent magnetic responsiveness biocompatibility of dye is constructed primitive alternate cycles to be soaked in a polyelectrolyte solution and b polyelectrolyte solution being grafted with supermolecule donor, described b polyelectrolyte can be interacted by electrostatic, hydrogen bond, coordinate bond etc. with a polyelectrolyte to carry out the polyelectrolyte of LbL, and cycle alternation soaks can construct the certain thickness polyelectrolyte multilayer film containing this supermolecule donor of primitive finishing in green.Simultaneously, red magnetic responsiveness biocompatibility is had to construct primitive alternate immersion in the c polyelectrolyte solution being grafted with Supramolecular Receptors and d polyelectrolyte solution dye, described d polyelectrolyte can be interacted by electrostatic, hydrogen bond, coordinate bond etc. with c polyelectrolyte to carry out the polyelectrolyte of LbL, and cycle alternation soaks can construct the certain thickness polyelectrolyte multilayer film containing this Supramolecular Receptors of primitive finishing in redness.
Equally, the polyelectrolyte being grafted with biological identification molecule can by step 3) modify and construct primitive surface to magnetic responsiveness biocompatibility.
Magnetic responsiveness biocompatibility is constructed primitive and is changed to substrate, with above-mentioned steps 1) to step 3) identical, supermolecule can be modified at substrate surface.
4. Magnetic guidance is modified with supramolecular magnetic responsiveness biocompatibility and constructs primitive and construct ordered 3 D structure
1) in the culture dish having certain water gaging, placement is modified with supramolecular substrate, is modified with supramolecular magnetic responsiveness biocompatibility constructs primitive in water surface dispersion;
2) local magnetic field is applied with Magnet or Three-Dimensional Magnetic operating means, magnetic responsiveness biocompatibility is made to construct primitive response external magnetic field, can move along with the movement in magnetic field or change, magnetic responsiveness biocompatibility is constructed primitive and is positioned to assigned address by magnetic manipulation field;
3) slowly absorb water to reduce water level, make to be modified with supramolecular magnetic responsiveness biocompatibility to construct primitive and be modified with supramolecular substrate surface and fully contact, reach intermolecular interaction distance, after a period of time, this magnetic responsiveness biocompatibility construct primitive can by the supermolecule of its finishing to (be subject to) body be modified with supermolecule by (to) substrate of body to be interacted by supermolecule and fixes, the supermolecule that the supermolecule namely constructing primitive finishing is modified to (being subject to) body molecule and substrate surface is subject to (to) interaction of body molecule generation supermolecule, this magnetic responsiveness biocompatibility is constructed after primitive is fixed, by the impact no longer by changes of magnetic field,
Supermolecule described in the present invention refers to the effect both sides that a kind of supermolecule interaction (such as electrostatic interaction) relates to receptor.
4) again add water, modify supermolecule with induced by magnetic field second and construct primitive to the magnetic responsiveness biocompatibility of (being subject to) body, magnetic is carried to assigned address, namely fixing with first magnetic responsiveness biocompatibility constructs that primitive is rectangular to be parallel to each other and the mutually concordant position of end points, the two spacing can regulate within the scope of one millimeter at ten microns, according to above-mentioned steps 3) continue location and fix, required magnetic responsiveness biocompatibility can be constructed construct primitive pattern in substrate, form ground floor magnetic responsiveness biocompatibility and construct primitive;
5) similar step 3) and 4), be modified with supermolecule with induced by magnetic field and construct elementary motions to the magnetic responsiveness biocompatibility of (being subject to) body, magnetic is carried to the magnetic responsiveness biocompatibility that ground floor assembled and constructs above primitive, make this be modified with supermolecule to construct primitive to the magnetic responsiveness biocompatibility of (be subject to) body and to be interacted by supermolecule and be fixed on the magnetic responsiveness biocompatibility that ground floor assembled and construct on primitive, formation second layer magnetic responsiveness biocompatibility constructs primitive;
6) ordered 3 D structure of the required number of plies is constructed from bottom to up.
5. the magnetic responsiveness biocompatibility that Magnetic guidance is modified with the molecule of supermolecule and/or bio-identification effect is constructed primitive and is constructed the ordered 3 D structure at specific site with the particular growth factor
1) for different somatomedin (belonging to protein matter), first, by 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride/N-hydroxysuccinimide, i.e. EDC/NHS catalyst system and catalyzing, respectively specific recognition site labelling is carried out to somatomedin in aqueous, as biotin (biotin) labelling and bacillus cereus rna enzyme inhibitor (barstar) labelling;
2) part magnetic responsiveness biocompatibility is constructed primitive and modify supermolecule, part magnetic responsiveness biocompatibility is constructed primitive and is modified supermolecule and the molecule (biotin or barstar) with bio-identification effect simultaneously;
3) in the culture dish having certain water gaging, placement is modified with supramolecular substrate, is modified with supramolecular magnetic responsiveness biocompatibility constructs primitive in water surface dispersion;
4) local magnetic field is applied with Magnet or Three-Dimensional Magnetic operating means, make to be modified with supramolecular magnetic responsiveness biocompatibility and construct primitive response external magnetic field, can move along with the movement in magnetic field or change, any one magnetic responsiveness biocompatibility is constructed primitive and is moved to and be modified with optional position above supramolecular substrate by magnetic manipulation field;
5) slowly absorb water to reduce water level, make to be modified with supramolecular biocompatibility to construct primitive and be modified with supramolecular substrate surface and fully contact, reach intermolecular interaction distance, after a period of time, this be modified with supramolecular magnetic responsiveness biocompatibility construct primitive can by the supermolecule on its surface to (be subject to) body be modified with supermolecule by (to) substrate surface of body to be interacted by supermolecule and fixes, the supermolecule that the supermolecule namely constructing primitive finishing is modified to (being subject to) body molecule and substrate surface is subject to (to) interaction of body molecule generation supermolecule, this magnetic responsiveness biocompatibility is constructed after primitive is fixed, by the impact no longer by changes of magnetic field,
6) again add water, operate second with magnetic field to be modified with supramolecular magnetic responsiveness biocompatibility and to construct primitive, magnetic is carried to assigned address in substrate, namely fixedly construct primitive to be parallel to each other and the mutually concordant position of end points with first, the two spacing can regulate within the scope of one millimeter at ten microns, according to above-mentioned steps 5) continue location and fix, form ground floor magnetic responsiveness biocompatibility parallel to each other, periodic arrangement and construct primitive;
7) similar step 5) and 6), the magnetic responsiveness biocompatibility being simultaneously modified with supermolecule and bio-identification effect molecule (biotin) with induced by magnetic field constructs elementary motions, magnetic is carried to ground floor magnetic responsiveness biocompatibility and constructs above primitive, its placement location has following characteristics: constructing primitive with ground floor has intersection point, and all angles constructed between primitive of ground floor can regulate between 0-90 degree.Make this magnetic responsiveness biocompatibility construct primitive to be interacted by supermolecule and be fixed on ground floor magnetic responsiveness biocompatibility and construct on primitive;
8) similar step 7), the magnetic responsiveness biocompatibility being simultaneously modified with supermolecule and another kind of bio-identification effect molecule (barstar) with induced by magnetic field constructs primitive to same layer another location, or the desired location of other layer;
9) construct the ordered 3 D structure of the required number of plies from bottom to up, and construct primitive in desired location introducing with the biocompatibility of specific biological recognition site;
10) by step 9) prepared by ordered 3 D structure be soaked in can and biotin, protein mixed solution (the i.e. Avidin avidin of barstar specific recognition, bacillus cereus rna enzyme barnase) in, make that the bio-identification site in ordered 3 D structure can (biotin/avidin identifies respectively by respective specific biological combination, barstar/barnase identifies), identify corresponding somatomedin, the orientation realizing different somatomedin in ordered 3 D structure is introduced, for further cell differentiation provides complicated chemistry, biological three-dimensional environment,
11) for prepared ordered 3 D structure, Cytotoxic evaluation is carried out by MTT standard method, be trained fibrocyte, fat stem cell, endotheliocyte, mescenchymal stem cell etc. and carry out cell adsorption experiment, the three-dimensional rack being adsorbed with mescenchymal stem cell is cultivated for a long time, investigates the differentiation situation of stem cell under introduced somatomedin effect.
Described biocompatibility is constructed primitive and is comprised PDMS, hydrogel, polylactic acid, polylactic acid-glycol acid, polycaprolactone, collagen, chitosan etc.
Described macroscopical Supramolecular Assembling yardstick comprises ten microns to a centimeter scale scope.
The three-dimensional order tissue engineering bracket of macroscopical Supramolecular Assembling prepared by the present invention is stacking shape ordered 3 D structure, described ordered 3 D structure by multiple at two-dimensional directional abreast, periodically the primitive of constructing of ordered arrangement form, two primitives of constructing contacted with each other realize two surperficial connections by supermolecule interaction the occurring to (be subject to) body functional group of the upper complementation of modifying in surface separately; The size of described support is 10
-5~ 10
-2m.
Beneficial effect of the present invention is as follows:
Macroscopical Supramolecular Assembling provided by the invention prepares the method for three-dimensional order tissue engineering bracket, obtain size by multiple micro-processing method and construct primitive at the biocompatibility of ten microns to a centimetre, and construct at biocompatibility in the preparation of primitive and introduce magnetic nano-particle to make to construct primitive response external magnetic field.Construct primitive to prepared biocompatibility to carry out finishing and obtain with the complementary functional groups of supermolecular mechanism as the complementary functional groups of Host-guest Recognition, electrostatic, hydrogen bond, coordination and bio-identification effect, make the above-mentioned complementary biological compatibility construct primitive and can be interacted by supermolecule and realize macroscopical Supramolecular Assembling and be fixed up.And then, utilize external magnetic field to construct primitive to biocompatibility in three dimensions accurately to locate, and construct primitive in the supermolecule interaction fixed biologically compatibility that assigned address utilizes operating platform substrate and biocompatibility to construct between primitive, thus progressively build ten microns of ordered 3 D structures to a cm size scope from bottom to top; Construct in process at three dimensional structure, construct primitive surface biological recognition site by biocompatibility and directedly can introduce appointment somatomedin, thus realize the orientation introducing of multiple somatomedin in three-dimensional stent material inside.The method can provide a kind of preparation newly to have the method for complicated chemical, bioenvironmental ordered 3 D structure, be a kind of there is universality, gentle, biocompatible preparation method, be expected to the masterplate for the preparation of Cell differentiation inducing activity and three-dimensional tissue's engineering rack.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1 a and Fig. 1 b illustrates the transmission electron microscope photo of synthesis ferriferrous oxide nano-particle;
Fig. 2 a illustrate synthesis pure ferriferrous oxide nano-particle and
Fig. 2 b illustrates that magnetic responsiveness constructs the hysteresis curve of primitive;
Fig. 3 illustrates square acrylic masterplate and mechanical grip optical photograph;
Fig. 4 illustrates that magnetic responsiveness PDMS cube constructs primitive;
Fig. 5 illustrates that primitive constructed by magnetic responsiveness hydrogel;
Fig. 6 illustrates that primitive schematic flow sheet is constructed in Frozen sections preparation;
Fig. 7 illustrates that PDMS strip prepared by Frozen sections constructs primitive;
Fig. 8 illustrates that micro-embossing legal system constructs primitive schematic diagram for micron order;
Fig. 9 a, Fig. 9 b, Fig. 9 c and Fig. 9 d illustrate SU8 photoresist array prepared by Ultraviolet lithography;
Figure 10 a and Figure 10 b illustrates that primitive fluorescence photo constructed by the standby micron order hydrogel of micro-embossing legal system
Figure 10 c and Figure 10 d illustrates that the standby micron order hydrogel of micro-embossing legal system is constructed in primitive water and peels off ordinary optical photo;
Figure 11 a illustrates sintetics PAA-CD nuclear magnetic spectrogram;
Figure 11 b illustrates sintetics PAA-Azo nuclear magnetic spectrogram;
Figure 12 illustrates that alternately stratiform self-assembling technique method is modified and constructs primitive surface schematic flow sheet;
Figure 13 illustrates that Magnetic guidance macroscopic view Supramolecular Assembling constructs ordered 3 D structure schematic flow sheet;
Figure 14 a, Figure 14 b, Figure 14 c and Figure 14 d illustrate the ordered 3 D structure of Magnetic guidance macroscopic view constructed by Supramolecular Assembling;
Figure 15 a and Figure 15 b illustrates that specific site has the ordered 3 D structure of the particular growth factor;
Figure 16 illustrates the Cytotoxic evaluation result of material;
Figure 17 a and Figure 17 b illustrates cell adsorption experimental result.
Detailed description of the invention
In order to be illustrated more clearly in the present invention, below in conjunction with preferred embodiments and drawings, the present invention is described further.Parts similar in accompanying drawing represent with identical Reference numeral.It will be appreciated by those skilled in the art that specifically described content is illustrative and nonrestrictive, should not limit the scope of the invention with this below.
1. hydro-thermal method prepares ferroferric oxide magnetic nano-particles
1), by Iron(III) chloride hexahydrate be dissolved in ethylene glycol, stir and form clear yellow solution.
2), add sodium acetate and Polyethylene Glycol, continue vigorous stirring 30 minutes, form colour of loess color cloud liquid, join in politef reactor, sealing, heat 10 hours at 200 degrees celsius.
3), room temperature is cooled to, with the ferriferrous oxide nano-particle that washing with alcohol obtains, wash three times, 60 degrees Celsius of vacuum oven 6 hours, obtain black powder, its size and hysteresis curve are respectively as shown in Fig. 1 a and Fig. 1 b, Fig. 2 a and Fig. 2 b.
2.1 mechanical micro Process masterplate casting methods are prepared grade magnetic responsiveness biocompatibility and are constructed primitive
Biocompatibility for millimeter and above yardstick constructs primitive, prepared by the mode can processing masterplate cast molding by traditional mechanical.
1) adopt the laser-engraving technique in mechanical processing industry, polymethyl methacrylate (acrylic) sheet material is engraved required hollow pattern, as the masterplate preparing biocompatibility and construct primitive.By means of computer aided technique, on masterplate pattern shape, size is all easy carries out controlling and regulating at millimeter and above yardstick.For square pattern, the masterplate of primitive can be constructed as casting cube biocompatibility, as shown in Figure 3;
2) clean masterplate successively with ethanol, deionized water, after drying, be fixed on smooth glass surface;
3) at room temperature, the liquid biological compatibility is constructed primitive (for PDMS, the mixture of its performed polymer and cross-linking agent) fully mix dispersion with prepared ferriferrous oxide nano-particle, obtained mixture is filled in fixing masterplate square hole on a glass;
4) another glass plate is withheld up, and by glass plate-masterplate-glass plate three clamping, in 65 degrees Celsius of baking ovens, heat a period of time, solidification, the cube PDMS fritter that the demoulding obtains discrete magnetic responsiveness constructs primitive as biocompatibility, as shown in Figure 4.
Hydrogel biocompatibility is constructed to the preparation of primitive, same step can be adopted, by the liquid after hydrogel monomer, cross-linking agent, initiator mix homogeneously, fully dispersion is mixed with magnetic nano-particle, be filled in masterplate, after forming gel by thermal initiation or light initiation polymerization, the demoulding, as shown in Figure 5.For low melting materials such as polylactic acid, polylactic acid-glycol acid, polycaprolactone, collagen, chitosans, melt liquid can be become at relatively high temperatures, water after mixing with magnetic nano-particle and cast from metal matrix, be then cooled to cold curing, the demoulding.
2.2 Frozen sections are prepared micro-meter scale magnetic responsiveness biocompatibility and are constructed primitive
1), by microscope slide Piranha washing liquid (concentrated sulphuric acid: hydrogen peroxide=3:1v/v) clean, deionized water rinsing, nitrogen dries up.In hermetic container, drip a small amount of 1H, 1H, 2H, 2H-perfluoro capryl triethoxysilane, discrete placement clean microscope slide, at 60 c by this perfluor silane of vapour deposition on microscope slide, make microscope slide present hydrophobicity.
2), as shown in Figure 6, for PDMS, first PDMS performed polymer and cross-linking agent thereof, magnetic nano-particle are mixed with certain proportion, drip on prepared hydrophobic microscope slide, two ends mat thickness is the coverslip of 150 microns, and top presses hydrophobic microscope slide, forms hydrophobic microscope slide-PDMS mixture-hydrophobic microscope slide sandwich, be heating and curing under 65 degrees Celsius, the demoulding obtains the PDMS thin slice that thickness is 150 microns.
3), under the microscope, the PDMS thin slice of above-mentioned 150 microns is cut into the thin slice that width is 2 millimeters × 2 millimeters × 150 microns.
4), with the coated above-mentioned thin slice of freezing embedding medium OCT, setting freezing microtome step distance, the thin slice embedded is placed on the position perpendicular to the freezing microtome edge of a knife, cut under the control of stepper motor, can obtain being of a size of 2 millimeters × 150 microns × 10 ~ 100 microns rectangular in batches, wherein 10 ~ 100 microns for can set adjustable step distance, as shown in Figure 7.
2.3 micro-embossing legal systems construct primitive for micro-meter scale magnetic responsiveness biocompatibility
Micro-embossing method flow process is as shown in Figure 8:
1), rectangular for 200 microns × 25 microns × 20 microns hydrogels, prepare the array of SU8 photoresist at silicon chip surface with Ultraviolet lithography, as shown in Figure 9, Fig. 9 a is strip SU8 photoresist array, Fig. 9 b is one of them rectangular enlarged drawing and corresponding size constructing primitive, Fig. 9 c is the rectangular cross-sectional view constructing primitive, and Fig. 9 d is the size of rectangular cross-sectional view in Fig. 9 c.
2), on SU8 photoresist array the mixture of spin coating PDMS performed polymer and cross-linking agent thereof, above-mentioned array is copied, obtains the strip hole of opposite pattern, as masterplate.
3), by hydrogel monomer, cross-linking agent, initiator and viscosifier mix, and dispersed magnetic nanoparticle, this mixture is dropped on above-mentioned PDMS masterplate, and covers polyethylene terephthalate (PET) thin slice, compress.
4), by PDMS-hydrogel monomer mixture-PET sandwich by nanometer hot pressing instrument compress, fully can fill long strip type hole PDMS on, simultaneously removal overflow and remain in the liquid between masterplate and PET.
5), by 365nm ultraviolet lighting or add that thermal initiation hydrogel is crosslinked, solidification.Open PET, the surface energy rectangular due to surface energy and the gel of PET is mated more, so open in PET process, the gel piece that solidified is known from experience and is adhered on PET, can come off and to collect discrete hydrogel rectangular, as Figure 10 a in water, shown in Figure 10 b, Figure 10 c and Figure 10 d.
Constructing the functional group of primitive finishing in order to distinguish magnetic responsiveness biocompatibility, a small amount of dyestuff can be mixed to distinguish in mixing material.
3. magnetic responsiveness biocompatibility constructs the functional group of primitive finishing with supermolecular mechanism
1) the supramolecular polyelectrolyte that is modified with for replacing stratiform package technique synthesizes: adopt graft reaction or free-radical polymerized mode, supermolecule cyclodextrin and diphenyl diimide are introduced in polyacrylic acid (PAA) this polyelectrolyte, obtain polyacrylic acid-cyclodextrin (PAA-CD), polyacrylic acid-diphenyl diimide (PAA-Azo), nuclear-magnetism figure as shown in figures 1 la and 1 lb.
2) magnetic responsiveness biocompatibility is constructed primitive (for PDMS cube fritter) to be cleaned by the mode that ethanol, deionized water are ultrasonic.
3) polyelectrolyte solution of preparation alternately needed for layered self-packaging (LbL) process: polyacrylic acid-cyclodextrin (PAA-CD), polyacrylic acid-diphenyl diimide (PAA-Azo): 0.1-1.0mg/mL.
4) as shown in Figure 12, a kind of biocompatibility is constructed primitive (dying green) alternate cycles to be soaked in poly-(diallyldimethylammonium chloride) (PDDA) and PAA-CD solution, obtain certain thickness PDDA/PAA-CD multilayer film; Another kind of biocompatibility is constructed primitive (dying redness) alternate cycles to be soaked in PDDA and PAA-Azo solution, obtain certain thickness PDDA/PAA-Azo multilayer film.Like this, biocompatibility constructs the surperficial supermolecule can modified with cyclodextrin or diphenyl diimide of primitive.
In addition, PAH-biotin can be become at PAH (PAH) side chain graft biotin (biotin), formed containing a kind of supramolecular PAH-biotin/PAA multilayer film of biotin by participating in above-mentioned alternately stratiform package technique, or simultaneously with cyclodextrin and the supramolecular PAH-biotin/PAA-CD multilayer film of biotin two kinds.Similarly, the polyelectrolyte PAH-barnase containing bacillus cereus rna enzyme can be introduced, obtain the multilayer film of PAH-barnase/PAA-CD.
Mix in pre-cast solution such as, with supramolecular compound, PAA-CD or PAA-Azo, also can realize supermolecule in finishing that prepared biocompatibility constructs primitive.
The finishing of ordered 3 D structure operating platform substrate (piezoid, sheet glass or silicon chip): first, by Piranha washing liquid (concentrated sulphuric acid: hydrogen peroxide=3:1v/v), piezoid or sheet glass or silicon chip are cleaned, deionized water rinsing, nitrogen dries up; Then piezoid or sheet glass or silicon chip alternate cycles are soaked in PDDA and PAA-CD solution, obtain certain thickness PDDA/PAA-CD multilayer film.
4. Magnetic guidance be modified with supramolecular magnetic responsiveness biocompatibility construct primitive assembling construct ordered 3 D structure
1) primitive is constructed for strip magnetic responsiveness PDMS biocompatibility, place in the culture dish having certain water gaging and be modified with at the bottom of the piezoid of PDDA/PAA-CD multilayer film or sheet glass or silicon wafer-based, be modified with the supramolecular PDMS of diphenyl diimide in water surface dispersion rectangular;
2) as shown in figure 13, local magnetic field is applied with Magnet or Three-Dimensional Magnetic operating means, make to be modified with the rectangular response external magnetic field of the supramolecular PDMS of diphenyl diimide, can move along with the movement in magnetic field or change, magnetic manipulation field will be modified with that the supramolecular PDMS of diphenyl diimide is rectangular is positioned to suprabasil optional position;
3) slowly absorb water to reduce water level, make to be modified with that the supramolecular PDMS of diphenyl diimide is rectangular fully to be contacted with operating platform substrate surface, reach intermolecular interaction distance, after a period of time, this PDMS bar can be interacted by supermolecule by the cyclodextrin supermolecule of the diphenyl diimide supermolecule on its surface and operating platform substrate surface and make to be modified with diphenyl diimide supramolecular PDMS bar and be fixed on suprabasil optional position, no longer by the impact of changes of magnetic field after it is fixing;
4) again add water, operating second with magnetic field, to be modified with the supramolecular PDMS of diphenyl diimide rectangular, magnetic is carried to rectangular with first PDMS in substrate being parallel to each other and the mutually concordant position of end points, the two spacing can regulate within the scope of one millimeter at ten microns, according to above-mentioned steps 3) continue location and fix, can construct in operating platform substrate and required be modified with the rectangular pattern of the supramolecular PDMS of diphenyl diimide, form ground floor magnetic responsiveness biocompatibility that is parallel, periodic arrangement and construct primitive;
5) again add water, the rectangular motion of the supramolecular PDMS of cyclodextrin is modified with induced by magnetic field, magnetic is carried to ground floor and is modified with the rectangular top of the supramolecular PDMS of diphenyl diimide, its placement location has feature: have intersection point with ground floor PDMS bar, and the angle between all PDMS bars of ground floor can regulate between 0-90 degree.According to this arrangement mode, be modified with rectangular being fixed on by supermolecular mechanism of the supramolecular PDMS of cyclodextrin and be modified with on the supramolecular PDMS layer of diphenyl diimide, form second layer magnetic responsiveness biocompatibility and construct primitive;
6) ordered 3 D structure of the required number of plies is constructed from bottom to up, for obtained ordered 3 D structure, it is all adjustable in three dimensions that its magnetic responsiveness biocompatibility constructs the position of primitive, spacing and the number of plies, as Figure 14 a, shown in Figure 14 b, Figure 14 c and Figure 14 d.
5. the magnetic responsiveness biocompatibility that Magnetic guidance is modified with the molecule of supermolecule and/or bio-identification effect is constructed primitive assembling and is constructed the ordered 3 D structure at specific site with the particular growth factor
1) to introduce two kinds of different somatomedin, VEGF (VEGF) and fibroblast growth factor (FGF) are example, first, by 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride/N-hydroxysuccinimide, i.e. EDC/NHS catalyst system and catalyzing, in aqueous biotin (biotin) labelling and bacillus cereus rna enzyme inhibitor (barstar) labelling are carried out to two kinds of growth factor VEGFs and FGF, obtain VEGF-biotin, FGF-barstar;
2) primitive is constructed for strip magnetic responsiveness PDMS biocompatibility, by rectangular for part PDMS modification cyclodextrin or diphenyl diimide supermolecule, modification cyclodextrin supermolecule and Avidin (avidin) the molecule while that part PDMS being rectangular, modification cyclodextrin supermolecule and bacillus cereus rna enzyme molecule while that part PDMS being rectangular, Avidin molecule and diphenyl diimide supermolecule while that part PDMS being rectangular, part PDMS is rectangular modifies bacillus cereus rna enzyme molecule and diphenyl diimide supermolecule simultaneously;
3) place in the culture dish having certain water gaging and be modified with at the bottom of the piezoid of PDDA/PAA-CD multilayer film or sheet glass or silicon wafer-based, be modified with the supramolecular PDMS of diphenyl diimide in water surface dispersion rectangular;
4) local magnetic field is applied with Magnet or Three-Dimensional Magnetic operating means, make to be modified with the rectangular response external magnetic field of the supramolecular PDMS of diphenyl diimide, can move along with the movement in magnetic field or change, magnetic manipulation field will be modified with that the supramolecular PDMS of diphenyl diimide is rectangular is positioned to suprabasil assigned address;
5) slowly absorb water to reduce water level, make to be modified with that the supramolecular PDMS of diphenyl diimide is rectangular fully to be contacted with operating platform substrate surface, reach intermolecular interaction distance, after a period of time, this is modified with the supramolecular PDMS bar of diphenyl diimide and can be interacted by supermolecule fixing, no longer by the impact of changes of magnetic field after it is fixing by the cyclodextrin supermolecule of the diphenyl diimide supermolecule on its surface and operating platform substrate surface;
6) again add water, operating second with magnetic field, to be modified with the supramolecular PDMS of diphenyl diimide rectangular, magnetic is carried to suprabasil assigned address, according to above-mentioned steps 5) continue location and fix, can construct in operating platform substrate and required be modified with the rectangular pattern of the supramolecular PDMS of diphenyl diimide, form ground floor magnetic responsiveness biocompatibility and construct primitive;
7) again add water, be modified with the rectangular motion of PDMS of cyclodextrin supermolecule and Avidin molecule with induced by magnetic field simultaneously, magnetic is carried to ground floor and is modified with the rectangular top of the supramolecular PDMS of diphenyl diimide, make rectangular being fixed on by supermolecule interaction of PDMS being modified with cyclodextrin supermolecule and Avidin molecule be modified with on the supramolecular PDMS layer of diphenyl diimide, form second layer magnetic responsiveness biocompatibility and construct primitive;
8) similar step 7), with induced by magnetic field simultaneously containing bacillus cereus rna enzyme molecule and the supramolecular PDMS of cyclodextrin rectangular, be fixed to same layer another location, or Magnetic guidance is simultaneously containing bacillus cereus rna enzyme molecule and the rectangular assigned address being fixed to adjacent layer of the supramolecular PDMS of diphenyl diimide, also can be that Magnetic guidance is simultaneously containing Avidin molecule and the rectangular assigned address being fixed to adjacent layer of the supramolecular PDMS of diphenyl diimide;
9) construct the ordered 3 D structure of the required number of plies from bottom to up, and construct primitive, in desired location introducing as shown in Figure 15 a and Figure 15 b with the PDMS biocompatibility of specific recognition site (as avidin, barstar);
10) by step 9) prepared by structure be soaked in the mixed solution of VEGF-biotin and FGF-barstar, make the avidin site in ordered 3 D structure and barnase site can respectively by the effect of biotin/avidin or barnase/barstar specific binding, identify corresponding VEGF-biotin and FGF-barstar somatomedin, the orientation realizing different somatomedin in ordered 3 D structure is introduced, for further cell differentiation provides complicated chemistry, biological three-dimensional environment;
11) for step 9) prepared by structure, carry out Cytotoxic evaluation by MTT standard method, result shows that material therefor does not have cytotoxicity (as shown in figure 16).Be trained fibrocyte, fat stem cell, endotheliocyte, mescenchymal stem cell etc. and carry out cell adsorption experiment, the three-dimensional rack being adsorbed with mescenchymal stem cell is cultivated for a long time, investigates the differentiation situation of stem cell under introduced somatomedin effect.Cell adsorption experimental result, as shown in accompanying drawing 17a and Figure 17 b, embodies good cell adsorption, migration, growth and differ entiation effect.
Obviously; the above embodiment of the present invention is only for example of the present invention is clearly described; and be not the restriction to embodiments of the present invention; for those of ordinary skill in the field; can also make other changes in different forms on the basis of the above description; here cannot give exhaustive to all embodiments, every belong to technical scheme of the present invention the apparent change of extending out or variation be still in the row of protection scope of the present invention.
Claims (10)
1. the three-dimensional order tissue engineering bracket of a macroscopical Supramolecular Assembling, it is characterized in that: described support is stacking shape ordered 3 D structure, described ordered 3 D structure by multiple at two-dimensional directional abreast, periodically the primitive of constructing of ordered arrangement form, there is supermolecule by the upper functional group of modifying in surface separately and interact and realize two surperficial connections in two primitives of constructing contacted with each other; The size of described support is 10
-5~ 10
-2m.
2. macroscopical Supramolecular Assembling prepares a method for three-dimensional order tissue engineering bracket, it is characterized in that, the method comprises:
Magnetic responsiveness biocompatibility constructs the preparation of primitive;
Magnetic responsiveness biocompatibility constructs primitive finishing functional group;
The magnetic responsiveness biocompatibility being modified with functional group is constructed primitive and is constructed ordered 3 D structure;
Described magnetic responsiveness biocompatibility constructs the preparation of primitive, is to be constructed in primitive to biocompatibility by the mode of mixing and doping to introduce magnetic nano-particle and construct primitive to prepare magnetic responsiveness biocompatibility.
3. method according to claim 2, is characterized in that, the preparation that described magnetic responsiveness biocompatibility constructs primitive comprises the steps:
The preparation of magnetic nano-particle;
Magnetic responsiveness biocompatibility constructs the preparation of primitive.
4. method according to claim 2, is characterized in that, described in be modified with functional group magnetic responsiveness biocompatibility construct the step that primitive constructs ordered 3 D structure and comprise:
Magnetic responsiveness biocompatibility constructs primitive finishing functional group;
The magnetic responsiveness biocompatibility that Magnetic guidance is modified with functional group is constructed primitive and is constructed magnetic responsiveness biocompatibility that ordered 3 D structure or Magnetic guidance be modified with functional group and construct primitive and construct the ordered 3 D structure in specific biological recognition site with the particular growth factor.
5. method according to claim 4, is characterized in that, described in be modified with functional group magnetic responsiveness biocompatibility construct the step that primitive constructs ordered 3 D structure and specifically comprise:
1) place in moisture container and be modified with the substrate of functional group, what be modified with functional group in water surface dispersion constructs primitive; Described substrate being selected from glass sheet, piezoid or silicon chip; The described substrate being modified with functional group is for being modified with supramolecular substrate; The described primitive of constructing being modified with functional group supramolecularly constructs primitive for being modified with;
2) to step 1) in container apply magnetic field, the primitive of constructing being modified with functional group of described water surface dispersion is positioned to the suprabasil assigned address being modified with functional group under the influence of a magnetic field;
3) to step 2) in container slowly absorb water, to reduce water level in container, the primitive of constructing being modified with functional group that the water surface is disperseed fully contacts with the substrate surface being modified with functional group, reaches intermolecular interaction distance, realizes fixing after 1 minute to 5 minutes;
4) to step 3) in container again add water, what be modified with functional group with induced by magnetic field second constructs primitive, is positioned to the suprabasil assigned address being modified with functional group under the influence of a magnetic field;
5) step 3 is repeated) and 4), substrate is constructed and required constructs primitive pattern;
6) what be modified with functional group with induced by magnetic field constructs primitive, is positioned to established ground floor under the influence of a magnetic field and constructs above primitive, repeatedly repeat the ordered 3 D structure that this step then can construct the required number of plies from bottom to top.
6. method according to claim 4, is characterized in that, the magnetic responsiveness biocompatibility that described Magnetic guidance is modified with functional group is constructed primitive and constructed the step having an ordered 3 D structure of the particular growth factor in specific biological recognition site and specifically comprise:
1) in aqueous specific biological recognition site labelling is carried out to somatomedin;
2) place in moisture container and be modified with the substrate of functional group, what be modified with functional group in water surface dispersion constructs primitive; Described substrate being selected from glass sheet, piezoid or silicon chip; The described substrate being modified with functional group is for being modified with supramolecular substrate; The described primitive of constructing being modified with functional group supramolecularly constructs primitive for being modified with;
3) to step 2) in container apply magnetic field, make the primitive of constructing being modified with functional group under the action of a magnetic field, be positioned to the suprabasil assigned address being modified with functional group;
4) to step 3) in container slowly absorb water, to reduce water level in container, the primitive of constructing being modified with functional group that the water surface is disperseed fully contacts with the substrate surface being modified with functional group, reaches intermolecular interaction distance, realizes fixing after 1 minute to 5 minutes;
5) to step 4) in container again add water, what be modified with functional group with induced by magnetic field second constructs primitive, is positioned to the suprabasil assigned address being modified with functional group under the influence of a magnetic field;
6) step 4 is repeated) and 5), substrate is constructed and required constructs primitive pattern;
7) what be modified with functional group with induced by magnetic field constructs primitive, is positioned to established ground floor under the influence of a magnetic field and constructs above primitive, repeatedly repeat the ordered 3 D structure that this step then can construct the required number of plies from bottom to top; Be modified with described in this step functional group construct primitive comprise be modified with supramolecular construct primitive or be modified with supermolecule and/or bio-identification effect molecule construct primitive;
8) by step 7) prepared by ordered 3 D structure be soaked in and step 1) in the complementation of specific biological recognition site protein mixed solution in, make the multiple functional group with bio-identification effect in ordered 3 D structure can respectively by respective specific biological combination, identify corresponding somatomedin, the orientation realizing different somatomedin in ordered 3 D structure is introduced.
7. method according to claim 2, is characterized in that: described magnetic responsiveness biocompatibility is constructed primitive and is of a size of 10
-5~ 10
-2m.
8. method according to claim 2, is characterized in that: described magnetic responsiveness biocompatibility is constructed primitive and prepared by mechanical micro Process masterplate casting method, frozen section method or micro-embossing method.
9. method according to claim 2, is characterized in that: described magnetic responsiveness biocompatibility is constructed primitive finishing functional group and modified by replacing stratiform package technique or surface grafting or monomer blend method; Described biocompatibility is constructed primitive and is comprised PDMS, hydrogel, polylactic acid, polylactic acid-glycol acid, polycaprolactone, collagen or chitosan; Described ordered 3 D structure is of a size of 10 μm of-1cm.
10. method according to claim 4, is characterized in that: described specific biological recognition site, and for having the bioactive sites of specific recognition capability, described specific biological recognition site is biotin labeling or bacillus cereus enzyme inhibitor labelling.
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| CN113968977A (en) * | 2021-10-29 | 2022-01-25 | 山东理工大学 | Precise assembly method and assembly for macro-functional construction elements in single-phase system |
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