CN104353123A - Non-homogeneous hydrogel and preparation method and application thereof - Google Patents
Non-homogeneous hydrogel and preparation method and application thereof Download PDFInfo
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- CN104353123A CN104353123A CN201410629511.XA CN201410629511A CN104353123A CN 104353123 A CN104353123 A CN 104353123A CN 201410629511 A CN201410629511 A CN 201410629511A CN 104353123 A CN104353123 A CN 104353123A
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Abstract
The invention discloses non-homogeneous hydrogel and a preparation method and an application thereof, and belongs to the field of biomedical materials. The non-homogeneous hydrogel comprises gelatinized continuous phases and gel microspheres which are distributed in the continuous phases. The preparation method comprises the following steps: firstly dispersing a water solution of a gel material into an oily solution and carrying out emulsification and gelatinization treatment to obtain the gel microspheres; and building the non-homogeneous hydrogel from the gel microspheres and the gel material. The hydrogel is non-homogeneous, and different materials or same materials with different properties can be adopted to load different cells or same cells with different densities, so as to build the hydrogel material in non-homogeneous distribution. The non-homogeneous hydrogel is beneficial to rebuilding of tissues or organs, has a wide application prospect in the field of tissue engineering, and can also be used for building a multi-cell limited contact three-dimensional co-culture system.
Description
Technical field
The invention belongs to field of biomedical materials, be specifically related to a kind of nonuniformity hydrogel and its preparation method and application.
Technical background
Because hydrogel can build the environment close with extracellular matrix, for the sticking of cell, to grow and propagation provides space, so hydrogel has good biocompatibility usually.Hydrogel, as a kind of important timbering material, is widely used and shows good Reparation and Reconstruction prospect, therefore having become one of emphasis of domestic and international biomedical material research and application in field of tissue engineering technology.
In existing report, hydrogel is substantially all the homogeneous hydrogel be prepared into after same material or different materials compound, be no matter that in composition or structure, the inside of these hydrogels is all uniformity, multiple composition and the labyrinth of this and natural tissues are diverse.Therefore from bionical angle, the defect of this composition and structure is difficult to realize to organize or the reproduction of organ dysfunction.
Summary of the invention
Inventor is by finding to have the following disadvantages in prior art to the comprehensive analysis of prior art:
1. traditional homogeneous hydrogel is all more single on the Nomenclature Composition and Structure of Complexes, different microenvironments can not be built for different cell, affect sticking, grow and breeding of cell, cause the deficiency of cell-secretion factor kind and quantity, thus make the extracellular matrix of reconstruct undesirable;
2. existing direct contact or mediate contact co-culture system can not take into account the interaction of influencing each other of variety classes cell and cell and material, have ignored and wherein all may have a negative impact to the differentiation-inducing of stem cell on the one hand.
For the problems referred to above, the invention provides a kind of nonuniformity hydrogel and Limited contact co-culture system.
The present invention is achieved through the following technical solutions:
A kind of nonuniformity hydrogel, the continuous phase comprising gelation and the gel micro-ball be distributed in described continuous phase.Described hydrogel is nonuniformity, and described continuous phase and microsphere can adopt identical gel rubber material, obtains the Inhomogeneous wave fields that material shape structure distribution is uneven; Also different gel rubber materials can be adopted, can the local microenvironment composition of different parts and structure in bionical natural tissues when adopting different gel rubber materials, obtain the Inhomogeneous wave fields that material composition is uneven with structure; Modification can also be carried out in the preparation as required to continuous phase, or employing prepares described nonuniformity hydrogel through the gel micro-ball of different modification, can obtain material property (as mechanical property, degradation property etc.) Inhomogeneous wave fields pockety.In addition, the mode adopting microsphere and continuous phase to coordinate can provide abundant interface in hydrogel inside, be convenient to the transmission of nutrient substance and metabolite on the one hand, the interaction that can give full play between allogenic cell influences each other with difference intercellular on the other hand, the growth of promotion cell, propagation also further secretion formation natural extracellular matrix.
Alternately, in above-mentioned nonuniformity hydrogel, described gel micro-ball is spherical or subglobular, and distribution of sizes is 30-1000 micron.The microsphere of this size range is adopted to be conducive to obtaining abundant interface.
Alternately, in above-mentioned nonuniformity hydrogel, the material forming described continuous phase and gel micro-ball is the gel rubber material with good biocompatibility.The gel rubber material of good biocompatibility is adopted to be conducive to the formation sticked, grow and organize of cell.
Alternately, in above-mentioned nonuniformity hydrogel, the precursor material of described gel rubber material has good aqueous solubility, is convenient to emulsifying balling-up in preparation process.
Alternately, in above-mentioned nonuniformity hydrogel, the material forming described continuous phase and gel micro-ball is environment sensitive type gel rubber material.Environment sensitive type gel rubber material is adopted to be convenient to carry out gelation process.
Alternately, in above-mentioned nonuniformity hydrogel, described environmental sensitivity gel rubber material is at least one in temperature sensing material, light-sensitive material or ion-sensitive material.Further, described temperature sensing material can be that collagen, PNIPAM and derivant isogel temperature thereof are lower than at least one in the material of 40 DEG C; Described light-sensitive material can be at least one in the photosensitive polysaccharide material of water solublity such as methacrylic anhydride or maleic anhydride modified derivatives of hyaluronic acids, chondroitin sulfate derivatives, pulullan polysaccharide derivant, light trigger can be 2-hydroxyl-4 '-(hydroxy ethoxy)-2-methyl phenyl ketone (I2959), 1-hydroxycyclohexylphenyl acetone (I184), one in 2,2-dimethoxy-phenylf 1-Phenylethanone .s (I651); Described ion-sensitive material can be alginic acid derivative.
Alternately, in above-mentioned nonuniformity hydrogel, the continuous phase periphery of having carried the gelation of gel micro-ball at described bag is also coated with the continuous phase that the continuous phase of at least one deck gelation or described bag carried the gelation of gel micro-ball and is distributed in again in the continuous phase of other layer of gel as gel cell.
Alternately, in above-mentioned nonuniformity hydrogel, described gel micro-ball and or continuous phase in also bag be loaded with cell.Can also adjust as required bag carry cell kind and or concentration, obtain cell category and or the uneven hydrogel material of CONCENTRATION DISTRIBUTION.The material of cell density Gradient distribution can also be obtained.
Alternately, in above-mentioned nonuniformity hydrogel, wrap the cell carrying two or more, the three-dimensional co-culture system of various kinds of cell Limited contact can be formed.A kind of cell can be loaded in described continuous phase in this co-culture system, all the other different types of cells are loaded in respectively in different gel micro-balls and (in a gel micro-ball, load allogenic cell); Also two or more cell can be loaded in respectively in different gel micro-balls and (in a gel micro-ball, load allogenic cell).In the directional induction research of stem cell, research worker has confirmed that the physical property such as three dimensional structure and mechanical strength of material can affect the differentiation destiny of stem cell, also recognized that the interaction between variety classes cell is can not be uncared-for, the three-dimensional Dual culture of various kinds of cell is one of effective means of this problem of research simultaneously.In existing report, the mode realizing Dual culture has two kinds, a kind of is directly contact, on timbering material, Dual culture is carried out again after directly mixing by different types of cell, another kind of is mediate contact, namely in different support, Dual culture in same media environment is inoculated after different cell respectively, as transwell cultivating system.Same, from bionical angle, be no matter directly contact or mediate contact all exists certain defect, can not simulate different cell in physiological environment has localized contact but situation about not mixing.By Limited contact co-culture system of the present invention, separated by different types of cell in same culture carrier, carry out Dual culture by means of only Limited contact, better can simulate different cell in physiological environment has localized contact but situation about not mixing.This system can be used for studying different seed cell influencing each other and effect to tissue engineering bracket material in the co-culture system of Limited contact, is specially adapted to study the differentiation-inducing condition of stem cell and Mechanism Discussion.
Alternately, the collagen described in said method can be animal skins, tendon, tail tendon are one in the collagen prepared of raw material; Described cell can be the CFU-GM such as mesenchymal stem cells MSCs, fat stem cell, or the one in the adult cell such as chondrocyte, fibroblast, endotheliocyte.
Present invention also offers a kind of method preparing above-mentioned nonuniformity hydrogel, comprise the following steps:
(1) by the aqueous dispersion of gel rubber material in oily solution, through emulsifying and gelation process, obtain gel micro-ball; (2) described gel micro-ball and gel rubber material are built into nonuniformity hydrogel.
Alternately, in above-mentioned preparation method, described step (1) is specially: in oily solution, add the surfactant that concentration expressed in percentage by volume is 0% ~ 10%, then under mechanical agitation, add gel rubber material solution, gel rubber material is dispersed in described oily solution and forms water in oil microsphere, while stirring two-phase mixtures liquid, select corresponding gelation condition to realize the gelation of microsphere.In described gel rubber material solution, the concentration of gel rubber material is preferably 3-50mg/mL.
Alternately, in above-mentioned preparation method, described step (2) is specially and is scattered in continuous phase solution medium by described gel micro-ball or is scattered in through modification or in the continuous phase medium of partial gelation process, then carries out gelation process formation nonuniformity hydrogel to above-mentioned mixed system.
Alternately, in above-mentioned preparation method, carrying out adding cell in gelation process forward direction mixed solution in described step (1) and/or step (2), after mixing, carrying out gelation process again, the nonuniformity hydrogel that bag has carried cell can be obtained.As optional, before adding cell, the pH value of mixed solution can be adjusted to 6.5-7.5.The density adding the cell in the mixed solution after cell is preferably 10
3-10
9individual/mL.
Alternately, in above-mentioned preparation method, the concrete mode adding cell in gel rubber material solution can be: gel rubber material pH value of solution is adjusted to 6.5-7.5, leaves standstill or centrifugal de-soak, obtains neutral gel material solution; Cell through counting is made into cell suspension by a small amount of culture medium or buffer, then joins in above-mentioned neutral gel material solution.Described culture medium can be the one in RPMI1640, MEM, α MEM, and described buffer can be the one in normal saline, PBS buffer, HEPES buffer.
Alternately, in above-mentioned preparation method, described oily solution can be one in methyl-silicone oil, paraffin wet goods mineral oil or olive oil, Oleum Ricini, Semen Maydis oil, Semen Allii Tuberosi wet goods vegetable oil or its mixing, and prioritizing selection viscosity is the oily solution of 20-200mPas.In oily solution, add the surfactant of 0%-10% (v/v), described surfactant can be the one in Tween-20, Tween-40, Tween-60, Tween-80, Span-20, Span-40, Span-60, Span-80, Triton X-100.
Alternately, in above-mentioned preparation method, in described step (1), aqueous phase solution (aqueous solution of gel rubber material) is 0.5%-40% with the ratio of oil-phase solution, preferred 1%-5%, with the speed Keep agitation 1-30min of 100-1500rpm, Aqueous dispersions is made to become microsphere.
Alternately, in above-mentioned preparation method, described gelation process can be at least one that intensification, photo-initiated crosslinking, enzyme-catalyzed cross-linking etc. become in gluing method.Such as: temperature sensing material is warming up to more than gelling temp from low temperature and keeps 10-45min; The irradiation under ultraviolet ray 10-360 second of light-sensitive material specific wavelength; Ion-sensitive material dropping bivalent cation solution, as calcium chloride, zinc chloride, magnesium chloride etc. realize plastic.
Alternately, in above-mentioned preparation method, before the described step (2) can also to step (1) in obtained gel micro-ball or celliferous gel micro-ball wash, be separated, remove oil phase.The separate mode that described separation can select centrifugal or filtration etc. conventional; Described washing step be specially by gel micro-ball obtained in step (1) or celliferous gel micro-ball culture medium or containing the buffer of 0%-0.1%Tween 80 in dispersion, rinse 2-4 time.Described culture medium can be the one in RPMI1640, MEM, α MEM, and described buffer can be the one in normal saline, PBS buffer, HEPES buffer.
Alternately, in above-mentioned preparation method, obtained nonuniformity hydrogel is scattered in continuous phase solution medium or is scattered in through modification or in the continuous phase medium of partial gelation process, then gelation process formation multilamellar nonuniformity hydrogel is carried out to above-mentioned mixed system.
Present invention also offers a kind of application of described nonuniformity hydrogel, it is characterized in that, use it for and make bionical tissue renovation material or for organizational project or be used as the carrier of seed cell or be used as the Limited contact Dual culture carrier of various kinds of cell.
All features disclosed in this description, or the step in disclosed all methods or process, except mutually exclusive feature and/or step, all can combine by any way.
Beneficial effect of the present invention:
Hydrogel of the present invention is nonuniformity, the tissue engineering bracket material with better bionical composition and structure is built with microsphere compound continuous phase, the hydrogel material of the non-uniform Distribution such as material category or performance, cell category or density can be built, be conducive to the reconstruction of tissue or organ, have broad application prospects in field of tissue engineering technology.Also can be used for building the three-dimensional co-culture system of various kinds of cell Limited contact, the biological effect of many cells system can be given full play to, realize good tissue repair and rebuild effect.Limited contact co-culture system construction method described in this co-culture system is simple, and be applicable to multiple different cell, realize the objects such as the multiple Limited contact of different cell, the controlled distribution of cell density, good condition can be provided, for abduction mechanism discussion and clinical application research lay the foundation for the Induction of committed differentiation research of the pluripotent cells such as mesenchymal stem cells MSCs.
Accompanying drawing illustrates:
Fig. 1 is the light microscopic photo of the collagen microsphere loading chondrocyte in embodiment 3 of the present invention;
Fig. 2 is size and the distribution statistics of the collagen microsphere loading chondrocyte in embodiment 3 of the present invention;
Fig. 3 is the laser co-focusing picture that the collagen microspheres loading chondrocyte in embodiment 3 of the present invention cultivates 1d outward;
Fig. 4 is size and the distribution statistics of the collagen microsphere loading mesenchymal stem cells MSCs in embodiment 5 of the present invention;
Fig. 5 is the laser co-focusing picture that the collagen microspheres loading mesenchymal stem cells MSCs in embodiment 5 of the present invention cultivates 3d, 7d, 14d and 21d outward;
Fig. 6 is the size and the distribution statistics that load the hyaluronate microspheres of mesenchymal stem cells MSCs in embodiment 5 of the present invention;
Fig. 7 is the release profiles of bovine serum albumin in microsphere collagen hydrogel in embodiment 9 of the present invention and block collagen hydrogel;
Fig. 8 loads the microsphere collagen hydrogel of chondrocyte and the laser co-focusing picture of block collagen hydrogel In vitro culture 3d, 7d and 14d in embodiment 10 of the present invention;
Fig. 9 is the process chart of the preparation method of nonuniformity hydrogel of the present invention.
Detailed description of the invention:
Preparation technology's flow process of the present invention is (as shown in Figure 9): gel rubber material fully dissolves (or making cell material mixed liquor with cell suspension), gelation process is carried out after dispersion in oily solution, again by obtained product cleaning process, obtain the microsphere loading cell; Microsphere redispersion is in the continuous phase solution of temperature sensing material or light-sensitive material, and gelation process forms nonuniformity hydrogel.
Wherein, when using temperature sensing material, cell material mixed liquor needs to mix under cryogenic; When using light-sensitive material, need to add light trigger in cell material mixed liquor; Dispersion treatment is under certain stirring condition, makes cell material mixed liquor be scattered in oily solution; After Keep agitation disperses a period of time, carry out gelation process microsphere crude product; Microsphere is obtained after repeatedly cleaning separation or isolated by filtration; Be scattered in the continuous phase solution of temperature sensing material or light-sensitive material by the microsphere loading cell, further gelation is built into nonuniformity hydrogel.
Again foregoing of the present invention is described in further detail by the following examples.But this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following example.Not departing from any amendment made within the spirit and principles in the present invention, and the equivalent replacement made according to ordinary skill knowledge and customary means or improvement, all should be included in protection scope of the present invention.Raw materials usedly in following examples all can to buy from the market.
Embodiment 1
(1) 200mL Oleum Brassicae campestris is got, add a small amount of Tween-60, under condition of ice bath, utilizing magnetic stirring apparatus to stir Oleum Brassicae campestris with the rotating speed of 1500rpm, is that the collagen solution of 15mg/mL is slowly added drop-wise to continuous stirring 20min in Oleum Brassicae campestris by 1mL concentration, above-mentioned aqueous phase-oil phase mixed liquor is warming up to 37 DEG C, and remain stirring 10min, after having stirred, mixed liquor is cleaned and is separated, remove oil phase, obtain the collagen microsphere that mean diameter is about 30 microns.
(2) collagen microsphere obtained in step (1) being scattered in concentration is in the collagen solution of 15mg/mL, the above-mentioned collagen solution containing microsphere is transferred to mould, be warming up to 37 DEG C and keep 30min, the nonuniformity collagen hydrogel of collagen continuous phase parcel collagen microsphere can be obtained.Gained hydrogel is cut open, under scanning electron microscope, can be observed, between material internal microsphere and continuous phase, there is a large amount of interface.
Embodiment 2
(1) get 4mL olive oil to obtain with 6mL Semen Maydis oil stirring and evenly mixing under the rotating speed of 800rpm and mix oil-phase solution, under agitation, hyaluronic acid (hyaluronic acid through the maleic anhydride modified) solution (hyaluronic acid concentration is 3mg/mL) that 4mL contains light trigger I184 is slowly added drop-wise in above-mentioned mixing oil-phase solution, continuous stirring 30min.Employing wavelength is 365nm, light intensity is 30mW/cm
2ultraviolet radiation 360s initiated polymerization, make hyaluronate microspheres gelation, and remain stir 10min; Mixed liquor cleaned and is separated, removing oil phase, obtain the hyaluronate microspheres that mean diameter is about 1000 microns.
(2) hyaluronate microspheres obtained in step (1) is scattered in concentration be 8mg/mL in maleic anhydride modified hyaluronic acid solution, the above-mentioned hyaluronic acid solution containing microsphere is transferred to mould, and employing wavelength is 365nm, light intensity is 30mW/cm
2ultraviolet radiation 360s initiated polymerization, can obtain hyaluronic acid continuous phase parcel hyaluronate microspheres nonuniformity hyaluronic acid gel.
Cell adhesion experiment display, embodiment 1 has good biocompatibility and good nutrition mass transfer ability with the nonuniformity hydrogel of gained in embodiment 2, and cell well can stick, sprawl, creep and grow on its surface.
Embodiment 3
The collagen microsphere of chondrocyte is loaded in preparation according to the following steps:
(1) get the collagen solution that concentration is 10mg/mL, regulate about pH to 7.0 with a small amount of 2mol/L sodium hydroxide solution in ice bath after, being diluted to collagen final concentration is 4.5mg/mL;
(2) by above-mentioned collagen solution in 4 DEG C of standing 2h, after bubble is sloughed, obtain neutral gum original solution;
(3) preprepared is in the chondrocyte of exponential phase of growth, counts and use MEM culture medium to be made into cell suspension, then to be joined in the above-mentioned neutral gum original solution of 1mL and to be uniformly dispersed, make cell density be 1 × 10
6individual/mL;
(4) selection viscosity is the methyl-silicone oil 72mL of 50mPas, does not add surfactant;
(5) under condition of ice bath, utilize magnetic stirring apparatus to stir methyl-silicone oil with the rotating speed of 500rpm, slowly above-mentioned chondrocyte collagen mixed liquor is added drop-wise in methyl-silicone oil, continuous stirring 20min;
(6) above-mentioned aqueous phase-oil phase mixed liquor is warming up to 37 DEG C, and remains stirring 30min;
(7) after having stirred, carry out centrifugal to mixed liquor, then supernatant liquid is toppled over;
(8) precipitation after using the PBS wash buffer of pH 7.4 centrifugal, the more centrifugal supernatant liquid that inclines, repeat this cleaning centrifugal process 2 times;
(9) finally the centrifugal precipitation obtained is the collagen microsphere loading chondrocyte, is scattered in MEM culture medium and is used for follow-up cultivation.
After the collagen microsphere optical microscope being scattered in culture medium is taken pictures, add up its size with image processing software, as shown in Figure 1, as shown in Figure 2, visible collagen Microsphere Size is 202.1 ± 55.7 microns to optical photograph in statistical result, and distribution is comparatively concentrated.After loading the collagen microsphere cultivation 1d of chondrocyte, confocal laser scanning microscope is carried out in sampling, and as shown in Figure 3, visible cell sticks result, propagation good.
Embodiment 4
The collagen microsphere of chondrocyte is loaded in preparation according to the following steps:
(1) get the collagen solution that concentration is 15mg/mL, regulate about pH to 7.0 with a small amount of 1mol/L sodium hydroxide solution in ice bath after, being diluted to collagen final concentration is 8.5mg/mL;
(2) by above-mentioned collagen solution in 4 DEG C of centrifugal 10min, getting bubble free liquids is neutral gum original solution;
(3) preprepared is in the mesenchymal stem cells MSCs cell of exponential phase of growth, counts and use α MEM culture medium to be made into cell suspension, then to be joined in the above-mentioned neutral gum original solution of 1mL and to be uniformly dispersed, make cell density be 5 × 10
7individual/mL;
(4) selection viscosity is the liquid paraffin 36mL of 100mPas, adds 0.5% (v/v) Span-80, stirs acquisition oil-phase solution with magnetic stirring apparatus with the rotating speed of 800rpm;
(5) under condition of ice bath, slowly above-mentioned mesenchymal stem cells MSCs collagen mixed liquor is added drop-wise in oil-phase solution, continuous stirring 30min;
(6) above-mentioned aqueous phase-oil phase mixed liquor is warming up to 37 DEG C, and remains stirring 15min;
(7) after having stirred, carry out centrifugal to mixed liquor, supernatant liquid is toppled over after falling and precipitation is scattered in α MEM culture medium;
(8) by the above-mentioned α MEM culture medium containing collagen microsphere through 100 tm screen net filtrations, and to rinse by the culture medium of more than 50mL;
(9) precipitation finally obtained is the collagen microsphere loading mesenchymal stem cells MSCs, is scattered in α MEM culture medium and is used for follow-up cultivation.
After the collagen microsphere optical microscope being scattered in culture medium is taken pictures, add up its size with image processing software, as shown in Figure 4, visible collagen Microsphere Size is 194.0 ± 85.2 microns to result, and distribution is comparatively concentrated.Load the collagen microsphere continuous culture 14d of mesenchymal stem cells MSCs, and respectively when cultivating 3d, 7d and 14d sampling carry out confocal laser scanning microscope, as shown in Figure 5, visible cell sticks result, propagation good, and has obvious Extracellular Matrix Secretion.
Embodiment 5
The hyaluronate microspheres of mesenchymal stem cells MSCs is loaded in preparation according to the following steps:
(1) get the hyaluronic acid of appropriate methacrylic acid anhydride modification, become concentration to be the solution of 20mg/mL with PBS buffer;
(2) in above-mentioned solution, add light trigger I2959, make its final concentration be 0.1%;
(3) preprepared is in the mesenchymal stem cells MSCs cell of exponential phase of growth, counts and use α MEM culture medium to be made into cell suspension, then to be joined in the above-mentioned hyaluronic acid solution of 1mL and to be uniformly dispersed, make cell density be 1 × 10
7individual/mL;
(4) selection viscosity is the liquid paraffin 54mL of 80mPas, adds 0.2% (v/v) Span-80, stirs acquisition oil-phase solution with magnetic stirring apparatus with the rotating speed of 650rpm;
(5) under continuous stirring, slowly above-mentioned mesenchymal stem cells MSCs hyaluronic acid mixed liquor is added drop-wise in oil-phase solution, continuous stirring 35min;
(6) adopt that wavelength is 365nm, light intensity is 30mW/cm
2ultraviolet radiation 60s initiated polymerization, make hyaluronate microspheres gelation, and remain stir 10min;
(7) after having stirred, carry out centrifugal to mixed liquor, supernatant liquid is toppled over after falling and precipitation is scattered in α MEM culture medium;
(8) by the above-mentioned α MEM culture medium containing microsphere through 100 tm screen net filtrations, and to rinse by the culture medium of more than 50mL;
(9) precipitation finally obtained is the hyaluronate microspheres loading mesenchymal stem cells MSCs.
After the hyaluronate microspheres optical microscope being scattered in culture medium is taken pictures, add up its size with image processing software, as shown in Figure 6, visible hyaluronate microspheres is of a size of 144.2 ± 53.4 microns to result, and distribution is comparatively concentrated.
Embodiment 6
Prepare nonuniformity collagen hydrogel according to the following steps:
(1) get the collagen solution that concentration is 10mg/mL, regulate pH to 7.0 with a small amount of 2mol/L sodium hydroxide solution in ice bath after, being diluted to collagen final concentration is 6.0mg/mL;
(2) by above-mentioned collagen solution in 4 DEG C of centrifugal 10min, getting bubble free liquids is neutral gum original solution;
(3) the collagen microsphere (such as embodiment 1 and embodiment 2) of different for previously prepared good loading cell or allogenic cell different densities is scattered in above-mentioned neutral gum original solution;
(4) the above-mentioned collagen solution containing microsphere is transferred to mould, is warming up to 37 DEG C and keeps 30min can obtain the nonuniformity collagen hydrogel loading local high density cell.
Embodiment 7
Prepare nonuniformity collagen-hyaluronic acid hydrogel soft osteocyte-mesenchymal stem cells MSCs Limited contact cultivating system altogether according to the following steps:
(1) with pH be 7.4 the maleic anhydride modified hyaluronic acid solution of PBS buffer, concentration is 25mg/mL, and adding light trigger I2959 to concentration is 0.1%;
(2) preprepared is in the chondrocyte of exponential phase of growth, counts and use MEM culture medium to be made into cell suspension, then to be joined in the above-mentioned hyaluronic acid solution of 1mL and to be uniformly dispersed, make cell density be 1 × 10
6individual/mL;
(3) microsphere (the collagen microsphere of such as embodiment 2 and the hyaluronate microspheres of embodiment 3) of different for previously prepared good loading mesenchymal stem cells MSCs density is scattered in above-mentioned hyaluronic acid solution;
(4) the above-mentioned hyaluronic acid solution containing microsphere is transferred to mould, employing wavelength 365nm, light intensity are 30mW/cm
2ultraviolet radiation 90s, the Limited contact Dual culture aquogel system loading chondrocyte and mesenchymal stem cells MSCs can be obtained.
Embodiment 8
Few two kinds are taken in the gel micro-ball prepared in embodiment 1-5, common distribution in continuous phase solution medium or common distribution in through modification or in the continuous phase medium of partial gelation process, again gelation process is carried out to above-mentioned mixed system, the nonuniformity hydrogel of bag year multiple gel micro-ball simultaneously in continuous phase can be obtained.Described gel micro-ball can have different materials to make, and also can wrap and carry different cells, also can have different cell bags and carry density.At continuous phase solution medium described in the forward direction of described gelation process or common distribution in adding cell through modification or in the continuous phase medium of partial gelation process, bag in continuous phase can also be obtained and carried the nonuniformity hydrogel of cell.
In this nonuniformity hydrogel structure, the modifying processing step of part hydrogel can separate with cell, thus avoids modification process on the impact of cell, thus can obtain the more excellent cell-hydrogel complex of performance.
Cell culture experiments shows, in the nonuniformity hydrogel being loaded with various kinds of cell, various cell is region all energy normal growths residing for separately, in good condition.
Embodiment 8
By the nonuniformity hydrogel redispersion that obtains in above-described embodiment 1,2,6,7,8 in continuous phase solution medium or common distribution in through modification or in the continuous phase medium of partial gelation process, again gelation process is carried out to above-mentioned mixed system, multilamellar nonuniformity hydrogel can be obtained.
Embodiment 9
Mass-transfer performance contrast test
With bovine serum albumin (BSA) for simulation substance, preparation method described in the embodiment of the present invention 1 is installed, BSA is added in collagen solution in step (1), prepare the collagen microsphere hydrogel (nonuniformity hydrogel) of loading BSA, adopt traditional collagen hydrogel preparation method to prepare the block hydrogel (BSA carries out gelation process after fully mixing with collagen solution and obtains) of collagen of loading BSA simultaneously.Two kinds of methods all adopt identical collagen concentration, identical gelling temperature, load the BSA of same concentrations, the microsphere hydrogel obtained and block hydrogel are immersed in PBS buffer respectively, the interval some time buffer that takes a morsel detects protein content wherein, and calculates the releasing ratio of BSA in hydrogel with this.
Mass transfer experiment comparing result as shown in Figure 7, therefrom can find faster than the BSA release in block hydrogel of BSA in collagen microsphere, show that microsphere hydrogel has better mass transfer effect, the balance of material concentration inside and outside hydrogel can be realized in the short period of time.
Embodiment 10
Chondrocyte In vitro culture contrast test
The collagen solution adopting same concentrations is raw material, preparation is mounted with the microsphere hydrogel of equal densities chondrocyte and block hydrogel respectively, adopting identical Cell culture invitro condition, when cultivating 3d, 7d and 14d, confocal laser scanning microscope being carried out to sample respectively.
(upper row is block hydrogel to chondrocyte In vitro culture comparative test result as shown in Figure 8, lower row is microsphere hydrogel), therefrom can find the prolongation along with incubation time, in collagen microsphere hydrogel, the propagation of chondrocyte is more obvious, cellular morphology and coherent condition are all better than block collagen hydrogel, show that collagen microsphere hydrogel is more conducive to propagation, the phenotype of chondrocyte.
The foregoing is only the preferred embodiments of the present invention, is only illustrative for the purpose of the present invention, and nonrestrictive; Those of ordinary skill in the art understand, and can carry out many changes in the spirit and scope that the claims in the present invention limit to it, amendment, and even equivalence is changed, but all will fall into protection scope of the present invention.
Claims (10)
1. a nonuniformity hydrogel, is characterized in that, the continuous phase comprising gelation and the gel micro-ball be distributed in described continuous phase.
2. nonuniformity hydrogel according to claim 1, is characterized in that, described gel micro-ball is spherical or subglobular, and distribution of sizes is 30-1000 micron.
3. nonuniformity hydrogel according to claim 1, is characterized in that, the material forming described continuous phase and gel micro-ball is the gel rubber material of the environment sensitive type with good biocompatibility.
4. nonuniformity hydrogel according to claim 3, is characterized in that, described environmental sensitivity gel rubber material is at least one in temperature sensing material, light-sensitive material or ion-sensitive material.
5. nonuniformity hydrogel according to claim 1, it is characterized in that, the continuous phase periphery of having carried the gelation of gel micro-ball at described bag is also coated with the continuous phase that the continuous phase of at least one deck gelation or described bag carried the gelation of gel micro-ball and is distributed in again in the continuous phase of other layer of gel as gel cell.
6. a preparation method for nonuniformity hydrogel as claimed in claim 1, is characterized in that, comprise the following steps:
(1) by the aqueous dispersion of gel rubber material in oily solution, through emulsifying and gelation process, obtain gel micro-ball; (2) described gel micro-ball and gel rubber material are built into nonuniformity hydrogel.
7. preparation method according to claim 6, it is characterized in that, described step (1) is specially: in oily solution, add the surfactant that concentration expressed in percentage by volume is 0% ~ 10%, then under mechanical agitation, add gel rubber material solution, gel rubber material is dispersed in described oily solution and forms water in oil microsphere, while stirring two-phase mixtures liquid, select corresponding gelation condition to realize the gelation of microsphere.
8. preparation method according to claim 6, it is characterized in that, described step (2) is specially: be scattered in by described gel micro-ball in continuous phase solution medium or be scattered in through modification or in the continuous phase medium of partial gelation process, then carries out gelation process formation nonuniformity hydrogel to above-mentioned mixed system.
9. preparation method according to claim 6, it is characterized in that, obtained nonuniformity hydrogel is scattered in continuous phase solution medium or is scattered in through modification or in the continuous phase medium of partial gelation process, then gelation process formation multilamellar nonuniformity hydrogel is carried out to above-mentioned mixed system.
10. an application for nonuniformity hydrogel according to claim 1, is characterized in that, uses it for make bionical tissue renovation material or for organizational project or be used as the carrier of seed cell or be used as the Limited contact Dual culture carrier of various kinds of cell.
Priority Applications (1)
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| CN106938058A (en) * | 2017-03-27 | 2017-07-11 | 四川大学 | A kind of preparation method and application of de- acellular collagen gels microsphere support |
| CN109642213A (en) * | 2016-03-01 | 2019-04-16 | 牛津大学创新有限公司 | It is loaded with the phase transfer of the bracket of loading |
| CN110029084A (en) * | 2019-04-12 | 2019-07-19 | 河海大学常州校区 | A kind of regulatable nonuniformity glucan 3D gel of partial cross-linking intensity, preparation method and applications method |
| CN114457001A (en) * | 2021-12-30 | 2022-05-10 | 广东粤港澳大湾区国家纳米科技创新研究院 | Scaffold material and preparation method and application thereof |
| CN114456929A (en) * | 2021-11-25 | 2022-05-10 | 浙江大学 | Device and method for constructing localized cell load model in three-dimensional scaffold |
| CN114469875A (en) * | 2022-03-04 | 2022-05-13 | 南京鼓楼医院 | Preparation method of frozen porous drug-loaded cell-loaded microspheres and application of frozen porous drug-loaded cell-loaded microspheres in preparation of drugs for treating acute liver failure |
| CN114457001B (en) * | 2021-12-30 | 2024-05-03 | 广东粤港澳大湾区国家纳米科技创新研究院 | Bracket material and preparation method and application thereof |
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| CN109642213A (en) * | 2016-03-01 | 2019-04-16 | 牛津大学创新有限公司 | It is loaded with the phase transfer of the bracket of loading |
| US11549097B2 (en) | 2016-03-01 | 2023-01-10 | Oxford University Innovation Limited | Phase transfer of a cargo laden scaffold |
| CN109642213B (en) * | 2016-03-01 | 2023-02-24 | 牛津大学创新有限公司 | Phase transfer with loaded scaffold |
| CN106938058A (en) * | 2017-03-27 | 2017-07-11 | 四川大学 | A kind of preparation method and application of de- acellular collagen gels microsphere support |
| CN110029084A (en) * | 2019-04-12 | 2019-07-19 | 河海大学常州校区 | A kind of regulatable nonuniformity glucan 3D gel of partial cross-linking intensity, preparation method and applications method |
| CN110029084B (en) * | 2019-04-12 | 2023-04-07 | 河海大学常州校区 | Heterogeneous glucan 3D gel with adjustable local crosslinking strength, and preparation method and application method thereof |
| CN114456929A (en) * | 2021-11-25 | 2022-05-10 | 浙江大学 | Device and method for constructing localized cell load model in three-dimensional scaffold |
| CN114457001A (en) * | 2021-12-30 | 2022-05-10 | 广东粤港澳大湾区国家纳米科技创新研究院 | Scaffold material and preparation method and application thereof |
| CN114457001B (en) * | 2021-12-30 | 2024-05-03 | 广东粤港澳大湾区国家纳米科技创新研究院 | Bracket material and preparation method and application thereof |
| CN114469875A (en) * | 2022-03-04 | 2022-05-13 | 南京鼓楼医院 | Preparation method of frozen porous drug-loaded cell-loaded microspheres and application of frozen porous drug-loaded cell-loaded microspheres in preparation of drugs for treating acute liver failure |
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