CN109091705B - A kind of three-dimensional porous rack and its preparation method and application - Google Patents
A kind of three-dimensional porous rack and its preparation method and application Download PDFInfo
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
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- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
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Abstract
The invention discloses a kind of three-dimensional porous racks, it is the composite biological material that degradable natural biologic material synthesizes high molecular material composition with degradable artificial；Three-dimensional porous rack have hole is uniform, is mutually communicated, the 3-D ordered multiporous structure of distribution rule, aperture be 80 μm~350 μm.The invention also discloses the preparation method and application of three-dimensional porous rack, the advantages of this method combines lotion microflow control technique and composite biological material.Three-dimensional porous rack of the invention has good biocompatibility and biological degradability, have the characteristics that aperture be suitable for, be mutually communicated, is degradable, is compressible, shaping, effective drainage, good space is provided for drug carrier and sustained release, cell growth and metabolism and tissue repair, the component ratio of composite material ensures that it has good compression and resilience performance as sponge with the hole of dense distribution, it can better adapt to uterine cavity or other need the form at preventing adhesions position, have the function that effective preventing adhesions.
Description
Technical field
The present invention relates to three-dimensional porous tissue engineering bracket material technical fields, and in particular to a kind of three-dimensional porous rack and
Preparation method and application.
Background technique
In utero adhesion (IUA), also referred to as Asherman syndrome, the damage of the basal layer as caused by a variety of causes
Wound, leads to uterine neck or the part or all of adhesion of uterine cavity.Its clinical manifestation is irregular menstruation, secondary infertility, repeatedly premature labor, stream
It produces, the even foetal death of other Averse pregnancy outcomes seriously affects the physical and mental health of women.Although through cervical adhesion resection
It (TCRA) is the current main method for treating Asherman's syndrom, but the recurrence rate of post-operation adhesion is still very high, serious Asherman's syndrom
High recurrence rate is up to 60% or more.Therefore, adhering to after effectivelying prevent separation again is still the significant challenge in its treatment.Find and
Developing suitable tissue engineering bracket is a kind of very promising method, and the three-dimensional porous branch standby based on lotion microfluidic control
Frame is of great significance in terms of preventing Asherman's syndrom.
Tissue engineering bracket is more and more applied and is explored in terms of preventing Asherman's syndrom.However hyalomitome sour water
The biomaterials such as gel, polytetrafluoroethylene (PTFE), submucous layer of small intestine (SIS), there are nonabsorables, degradation in vivo rate
Too fast, the disadvantages of lacking flexibility with enough mechanical strengths, is restricted its application.
In the prior art, application No. is the patents of invention of CN201410629406.6 to disclose one kind based on microflow control technique
High molecular polymer timbering material preparation method.This method using drop micro fluidic device generate a large amount of uniform drops or
Spheric granules is intensively scattered in the organic solvent dissolved with high molecular polymer using drop or particle as pore-foaming agent, preparation
High molecular polymer timbering materials that provide specific morphology, uniform pores.But the bracket of this method preparation is unfavorable for
Cell adhesion and proliferation, reason first is that the bracket use artificial synthesized high molecular material, material hydrophilic is poor, cellular affinity compared with
It is weak, and catabolite slant acidity easily causes aseptic inflammation to react.Reason is second is that the bracket is sharp twice in a preparation process
With micro fluidic device, the bracket being prepared is it is difficult to ensure that inside and outside perforation.And the hole size for being mutually communicated between hole and being suitable for
Exactly cell adhesion, proliferation and the important factor in order of migration.In addition, the bracket of this method preparation is limited only to micron order ruler
Very little spherical shape or threadiness is not appropriate for the actual demand for clinically requiring tissue engineering bracket to have various forms and size.
Summary of the invention
In view of the deficiencies of the prior art, one of the objects of the present invention is to provide a kind of three-dimensional porous racks, it has good
Good biocompatibility and biological degradability, it is good material-Cellular interfaces that good hydrophilic property, cellular affinity are strong, and can
To be easily adjusted component, pore size, porosity and shape, therefore it can preferably meet clinical demand.It is suitable with aperture
Preferably, be mutually communicated, be degradable, is compressible, shaping, effective drainage the characteristics of, which is drug carrier and sustained release, thin
Intracellular growth and metabolism and tissue repair provide good space.Further, the component ratio of composite material is divided with intensive
The hole of cloth ensures that it has good compression and resilience performance as sponge, can better adapt to uterine cavity or other are needed in advance
Prevent adhesion the form at position, actually reaches the effect of effective preventing adhesions and tissue repair.
The second object of the present invention is to provide a kind of preparation method of three-dimensional porous rack, the solidification side of the preparation method
Formula is more simple and convenient, and only needs to have easy to operate, at low cost using a micro fluidic device in a preparation process
Honest and clean, the characteristics of can be mass.
The third object of the present invention is to provide a kind of load medicine three-dimensional porous rack including above-mentioned three-dimensional porous rack.
The fourth object of the present invention is to provide a kind of marrow stromal cell (BMSCs) including above-mentioned three-dimensional porous rack
Co-culture three-dimensional porous rack.
The fifth object of the present invention is to provide a kind of above-mentioned three-dimensional porous rack and is preparing the application in medical instrument, institute
Medical instrument is stated for preventing Asherman's syndrom or other position adhesions and tissue repair.
One of achieving the object of the present invention can be reached by adopting the following technical scheme that:
A kind of three-dimensional porous rack, which is characterized in that the three-dimensional porous rack be degradable natural biologic material with
Degradable artificial synthesizes the composite biological material of high molecular material composition；The three-dimensional porous rack uniform, phase with hole
It mutually penetrates through, the three-dimensional porous structure of distribution rule, aperture is 80 μm~350 μm.
Further, the degradable natural biologic material synthesizes the weight ratio of high molecular material with degradable artificial
For 1~4:1~20.
Further, the degradable natural biologic material includes but is not limited to sodium alginate, collagen, chitosan, silk
The one or more of fibroin.
Further, the artificial synthesized high molecular material includes but is not limited to methacrylic anhydride gelatin (Gelma), gathers
The one or more of glycol diacrylate.
Further, the three-dimensional porous rack further includes photoinitiator, polyethylene glycol propylene glycol-polyethylene glycol
(F108), lauryl sodium sulfate (SDS)；Photoinitiator, polyethylene glycol propylene glycol-polyethylene glycol (F108), dodecyl
The weight ratio of sodium sulphate (SDS) and composite biological material is 1:2:2:10~50.F108 and SDS helps to reduce emulsion droplet
It is broken.
Further, the three-dimensional porous rack can be by using purpose and demand to be prepared into different shape and size.
Two achieved the object of the present invention can be reached by adopting the following technical scheme that:
A kind of preparation method of three-dimensional porous rack characterized by comprising
The preparation step of monodisperse emulsion drop: size tunable is prepared with Nei phase using micro fluidic device, foreign minister
Monodisperse emulsion drop is cleaned and is collected to monodisperse emulsion drop；Wherein, foreign minister is composite biological material；Foreign minister and
It is interior mutually immiscible；
The preparation step of emulsion droplet template: the monodisperse emulsion drop being collected into is stood, or monodisperse is newborn
Liquid drop is collected into and stands by using in purpose and the previously prepared mold of various shapes of demand, and monodisperse emulsion drop can be from
It is assembled into close-packed structure, ultimately forms uniform neat emulsion droplet template；Then emulsion droplet template is solidified
Processing；
The collection step of three-dimensional porous rack: collecting the emulsion droplet template after solidification and clean, removes emulsion droplet
Interior phase in template, obtains that hole is uniform, is mutually communicated, the three-dimensional porous rack of distribution rule.
Further, the interior phase is one or more of edible oil, methyl-silicone oil.
Further, in the preparation step of monodisperse emulsion drop, the flow velocity of foreign minister is 1~5mL/h；The flow velocity of interior phase is
0.1~0.3mL/h.
Further, in the preparation step of monodisperse emulsion drop, the flow velocity of foreign minister is 1mL/h, 1.5mL/h, 2mL/h,
One of 2.5mL/h, 3mL/h, 3.5mL/h, 4mL/h, 4.5mL/h, 5mL/h；The flow velocity of interior phase is 0.1mL/h, 0.15mL/
H, one of 0.2mL/h, 0.25mL/h, 0.3mL/h.
Further, in the preparation step of emulsion droplet template, time of repose is at least 1min.
Further, in the preparation step of emulsion droplet template, the detailed process of curing process are as follows: using ultraviolet light to cream
Liquid drop template carries out one-step solidification, and the one-step solidification time is 2~5min, and emulsion droplet template is then dipped in calcium chloride solution
In or cooling carry out secondary curing, 2~5min of secondary curing time.
Further, in the collection step of three-dimensional porous rack, the detailed process of cleaning are as follows: by the emulsion droplet after solidification
Template is immersed in dehydrated alcohol, is placed in ultrasound 15s in Ultrasound Instrument, and it is ultrasonic again to change to new dehydrated alcohol after taking-up, such as
Three times, the interior phase in emulsion droplet template is completely removed for this cleaning；Then material is impregnated in deionized water, is displaced more
Remaining dehydrated alcohol after dry, obtains that hole is uniform, be mutually communicated, distribution rule hole is uniform, is mutually communicated, distribution rule
Three-dimensional porous rack.
Further, in the collection step of three-dimensional porous rack, the detailed process of cleaning are as follows: by the emulsion droplet after solidification
Template is immersed in methylene chloride, impregnates twice, the interior phase in emulsion droplet template is completely removed, then uses alcohol by each 30min
Gradient displacement removal methylene chloride, obtains that hole is uniform, be mutually communicated, distribution rule hole is uniform, is mutually communicated, distribution rule
Three-dimensional porous rack.
Three achieved the object of the present invention can be reached by adopting the following technical scheme that:
A kind of load medicine three-dimensional porous rack, which is characterized in that including three-dimensional porous branch described in an object of the present invention
Frame, the three-dimensional porous rack load have drug.
Further, it is described carry medicine three-dimensional porous rack the preparation method is as follows: that three-dimensional porous rack is dipped in drug is molten
In liquid, keep drug fully dispersed into hole, taken out after standing overnight, can be obtained and carry medicine three-dimensional porous rack.
Four achieved the object of the present invention can be reached by adopting the following technical scheme that:
A kind of BMSCs cell co-cultivation three-dimensional porous rack, which is characterized in that including described in an object of the present invention
Three-dimensional porous rack co-cultures three-dimensional porous rack and BMSCs cell 1-2 weeks, to BMSCs cell in three-dimensional porous rack
It is sufficiently proliferated, grows in hole, can be obtained MSC cell and co-culture three-dimensional porous rack.
Five achieved the object of the present invention can be reached by adopting the following technical scheme that:
Three-dimensional porous rack described in a kind of an object of the present invention exists preparing the application in medical instrument, feature
In the medical instrument is for preventing Asherman's syndrom or other position adhesions and tissue repair.
Further, the medical instrument is three-dimensional porous rack；Either, the medical instrument is that load medicine is three-dimensional porous
Bracket, the medicine three-dimensional porous rack that carries includes the three-dimensional porous rack, and the three-dimensional porous rack load has drug；Or
Person is that the medical instrument is that BMSCs cell co-cultures three-dimensional porous rack, and the BMSCs cell co-cultures three-dimensional porous branch
Frame includes the three-dimensional porous rack, three-dimensional porous rack and BMSCs cell is co-cultured 1-2 weeks, to BMSCs cell three
It ties up and is sufficiently proliferated, grows in the hole of porous support, can be obtained BMSCs cell and co-culture three-dimensional porous rack.
The beneficial effects of the present invention are:
1, three-dimensional porous rack of the invention use with complementary characteristic degradable natural biologic material with it is degradable
Artificial synthesized high molecular material composition composite biological material, three-dimensional porous rack have hole is uniform, is mutually communicated, is distributed
The three-dimensional porous structure of rule, aperture are 80 μm~350 μm, which is mutually suitable for most people body cell size.It is three-dimensional
Porous support has good biocompatibility and biological degradability, and good hydrophilic property, cellular affinity are strong, are good materials-
Cellular interfaces have the characteristics that aperture is suitable for, porosity is high, are mutually communicated, are degradable, is compressible, shaping, effective drainage,
The porous support provides good space for drug carrier and sustained release, cell growth and metabolism and tissue repair.Further,
The component ratio of composite material ensures that it has good compression and resilience performance, energy as sponge with the hole of dense distribution
It better adapts to uterine cavity or other needs the form at preventing adhesions position, actually reach the work of effective preventing adhesions and tissue repair
With.
2, the preparation method combination lotion microflow control technique of three-dimensional porous rack of the invention and composite biological material is excellent
Point uses oil-in-water (O/W) single emulsion system for template, collects and arranges careful multilayer drop, and cleaning is after solidification to remove it
In oily phase, obtain hole distribution orderly, the three-dimensional porous rack that is mutually communicated.Present invention combination microflow control technique accurately may be used
Control property, the three-dimensional porous rack being prepared have the advantages that hole size is uniform, porosity is high, are conducive to implant after compression
It is interior, and suitable size, the hole configurations that is mutually communicated are that drug carrier and sustained release, cell growth and metabolism and tissue repair mention
For space.It, can also be by using purpose and demand to prepare respectively in addition, three-dimensional porous rack has the hole configurations that is mutually communicated
Kind specific shape and size are adapted with the position form of preventing adhesions, while being conducive to the sepage outflow of injury region.The present invention
The curing mode that the preparation method of the three-dimensional porous rack of offer is specially designed for composite biological material is more simple and convenient,
Preparing in stent procedures only need to be easy to operate, low in cost, can be mass using a micro fluidic device, and having can be square
Just the advantages of the component of control accurate bracket, aperture, porosity, shapes and sizes.
3, three-dimensional porous rack of the invention has preferable elasticity, support force and mechanical property, after implantation can with uterine cavity or
The uterine cavity of indefinite form is adapted after gradation adherence Separation, effectively separates sidewalls and palace bottom, be can be applied to prevent and be repaired
Asherman's syndrom can also be applied to the adhesion prevention and tissue repair at other positions such as abdominal cavity.
4, load medicine three-dimensional porous rack of the invention, which can be immersed directly in medical fluid, carries medicine.Three-dimensional porous rack is immersed in
In the FITC-BSA solution of 20ug/ml, detecting that it uploads rate is 30% or more, and the Cumulative release amount for monitoring week age is reachable
To 40%.The three-dimensional porous rack of load bFGF of the invention can prevent Asherman's syndrom after rat uterus damages, promote inner membrance
It repairs, improves tissue vascularization and cellularised, help to improve uterogestation function, can be applied to prevent and repair uterine cavity and is viscous
Even.
5, cell of the invention co-cultures three-dimensional porous rack and can co-culture with BMSCs cell, and BMSCs cell, which has, to be divided
It turns to endometrial cell, vascular cell, biologically active secretory molecule, adjust immune function, help to improve uterogestation
Function can be applied to prevent and repair Asherman's syndrom.
Detailed description of the invention
In Fig. 1, (a) is the structural schematic diagram of micro fluidic device, and I pipe is dispersed phase input terminal, and II pipe is output end, III
Pipe is observation tube；It (b) is the structural schematic diagram of round tube and square tube；It (c) is the pictorial diagram of micro fluidic device；It (d) is respectively two kinds
Construct the pictorial diagram of micro fluidic device: coflow formula apparatus structure, opposite-flushing type apparatus structure.
In Fig. 2, A is the pictorial diagram for meeting the three-dimensional porous rack of rat uterine cavity shape；B is radius * high (0.5*0.5cm)
Three-dimensional porous rack pictorial diagram；C is its electron microscope；D is to show that bracket is mutually communicated the electron microscope of structure.
In Fig. 3, (i, v) sham-operation group, (ii, vi) Natural re generation group, (iii, vii) three-dimensional porous rack group, (iv,
Viii the porous support group of bFGF) is carried.The HE colored graph that (a, c) is 1 week and 6 weeks, (b, d) is respectively its corresponding partial enlargement
Figure, black arrow show endometrial gland；It (e) is 1 week, the statistical chart of 6 weeks each group endometrium thickness；It (f) is 1 week, 6
The statistical chart of all each group endometrial gland quantity.
In Fig. 4, (i, v) sham-operation group, (ii, vi) Natural re generation group, (iii, vii) three-dimensional porous rack group, (iv,
Viii the porous support group of bFGF) is carried.(a, b) is 1 week, 6 weeks each group horse pine colored graphs；(c, d) is 1 week, 6 weeks new green bloods of each group
Pipe colored graph；It (e) is 1 week, 6 weeks each group fibrosis of uterus area statistics figures；It (f) is 1 week, 6 weeks each group nascent blood vessel density systems
Meter figure.
Specific embodiment
In the following, being described further in conjunction with specific embodiment to the present invention, it should be noted that is do not collided
Under the premise of, new embodiment can be formed between various embodiments described below or between each technical characteristic in any combination.
A kind of three-dimensional porous rack, which is characterized in that the three-dimensional porous rack be degradable natural biologic material with
Degradable artificial synthesizes the composite biological material of high molecular material composition；The three-dimensional porous rack uniform, phase with hole
It mutually penetrates through, the three-dimensional porous structure of distribution rule, aperture is 80 μm~350 μm.
As the mode that further carries out, the degradable natural biologic material synthesizes macromolecule with degradable artificial
The weight ratio of material is 1~4:1~20.
As the mode that further carries out, the degradable natural biologic material includes but is not limited to sodium alginate, glue
The one or more of original, chitosan, fibroin albumen.
As the mode that further carries out, the artificial synthesized high molecular material includes but is not limited to that methacrylic anhydride is bright
The one or more of glue (Gelma), polyethyleneglycol diacrylate.
As the mode that further carries out, the three-dimensional porous rack further includes photoinitiator, polyethylene glycol the third two
Alcohol-polyethylene glycol (F108), lauryl sodium sulfate (SDS)；Photoinitiator, polyethylene glycol propylene glycol-polyethylene glycol
(F108), lauryl sodium sulfate (SDS) and the weight ratio of composite biological material are 1:2:2:10~50.F108 and SDS are helped
In the broken of reduction emulsion droplet.
As the mode that further carries out, the three-dimensional porous rack can be by using purpose and demand to be prepared into not similar shape
Shape and size.
A kind of preparation method of three-dimensional porous rack characterized by comprising
The preparation step of monodisperse emulsion drop: size tunable is prepared with Nei phase using micro fluidic device, foreign minister
Monodisperse emulsion drop is cleaned and is collected to monodisperse emulsion drop；Wherein, foreign minister is composite biological material；Foreign minister and
It is interior mutually immiscible；
The preparation step of emulsion droplet template: the monodisperse emulsion drop being collected into is stood, or monodisperse is newborn
Liquid drop is collected into and stands by using in purpose and the previously prepared mold of various shapes of demand, and monodisperse emulsion drop can be from
It is assembled into close-packed structure, ultimately forms uniform neat emulsion droplet template；Then emulsion droplet template is solidified
Processing；
The collection step of three-dimensional porous rack: collecting the emulsion droplet template after solidification and clean, removes emulsion droplet
Interior phase in template, obtains that hole is uniform, is mutually communicated, the three-dimensional porous rack of distribution rule.
Referring to Fig.1, the assembling of micro fluidic device: whole device is in a piece of clean smooth glass slide over-assemble.First will
III pipe is placed in suitable position on glass slide, is then reversely inserted into I pipe and II pipe from the both ends of III pipe respectively, makes the point of I pipe
End gos deep into the inside of II pipe but should not be completely plugged by nozzle, or keeps the tip of I pipe opposite with II pipe, then under the microscope
Being adjusted to the relative position of two pipes, which makes its central axes overlap, is presented coaxial pattern of rows and columns.Finally pacify at III pipe both ends
Fill syringe needle.In assembling process fixation and linking in need place completed by quick-drying gelatin.It is finally assembling to
Shown in device such as Fig. 1 (c).I pipe, II pipe are all made of capillary glass tube, and for I pipe as input pipe, one end is smooth, with PE hose phase
It connects, is inputted for interior phase solution, the other end can get superfine contraction according to different experiments purpose by microelectrode controller
Port, and being cut into internal diameter using glass cutter is micron-sized port, the main function of the port is inside and outside using shrinking and making
Phase solution forms the drop microballoon smaller than internal diameter of the pipeline, change in size range is big by shearing force and liquid tension；II pipe one end
The micron order bore for being melt into slightly contraction state is heated using alcohol blast burner, another port smooth surface is used for liquid and lotion
The outflow of drop；III Guan Wei great round tube or rectangular tube are covered in two round tube intersections, are provided to the inflow of external solution empty
Between, and realize the real-time observation to droplet shearing situation.
Certainly, micro fluidic device of the invention can also use other micro fluidic devices in the prior art.The system of foreign minister
Preparation Method: each component is uniformly mixed.
As the mode that further carries out, the interior phase is one or more of edible oil, methyl-silicone oil.
As the mode that further carries out, in the preparation step of monodisperse emulsion drop, the flow velocity of foreign minister is 1~5mL/h；
The flow velocity of interior phase is 0.1~0.3mL/h.
As the mode that further carries out, in the preparation step of monodisperse emulsion drop, the flow velocity of foreign minister be 1mL/h,
One of 1.5mL/h, 2mL/h, 2.5mL/h, 3mL/h, 3.5mL/h, 4mL/h, 4.5mL/h, 5mL/h；The flow velocity of interior phase is
One of 0.1mL/h, 0.15mL/h, 0.2mL/h, 0.25mL/h, 0.3mL/h.
As the mode that further carries out, in the preparation step of emulsion droplet template, time of repose is at least 1min.
As the mode that further carries out, in the preparation step of emulsion droplet template, the detailed process of curing process are as follows: benefit
One-step solidification is carried out to emulsion droplet template with ultraviolet light, the one-step solidification time is 2~5min, then soaks emulsion droplet template
In calcium chloride solution or cooling carries out secondary curing, 2~5min of secondary curing time.
As the mode that further carries out, in the collection step of three-dimensional porous rack, the detailed process of cleaning are as follows: will solidify
Emulsion droplet template afterwards is immersed in dehydrated alcohol, is placed in ultrasound 15s in Ultrasound Instrument, new dehydrated alcohol is changed to after taking-up
Ultrasonic again, three times, the interior phase in emulsion droplet template is completely removed for so cleaning；Then material is immersed in deionized water
In, it displaces extra dehydrated alcohol, after dry, obtains that hole is uniform, is mutually communicated, the three-dimensional porous rack of distribution rule.
As the mode that further carries out, in the collection step of three-dimensional porous rack, the detailed process of cleaning are as follows: will solidify
Emulsion droplet template afterwards is immersed in methylene chloride, impregnates twice, each 30min will be interior mutually complete in emulsion droplet template
Removal, then methylene chloride is removed with the displacement of alcohol gradient, obtain that hole is uniform, is mutually communicated, the three-dimensional porous branch of distribution rule
Frame.
A kind of load medicine three-dimensional porous rack, including three-dimensional porous rack described in an object of the present invention, the three-dimensional
Porous support load has drug.
As the mode that further carries out, it is described carry medicine three-dimensional porous rack the preparation method is as follows: by three-dimensional porous branch
Frame is dipped in drug solution, keeps drug fully dispersed into hole, is taken out after standing overnight, and be can be obtained and is carried the three-dimensional porous branch of medicine
Frame.
A kind of BMSCs cell co-cultivation three-dimensional porous rack, including three-dimensional porous branch described in an object of the present invention
Frame, the three-dimensional porous rack load have cell.Three-dimensional porous rack and BMSCs cell are co-cultured 1-2 weeks, it is thin to BMSCs
Born of the same parents are sufficiently proliferated in the hole of three-dimensional porous rack, grow, and can be obtained BMSCs cell and co-culture three-dimensional porous rack.
Three-dimensional porous rack described in a kind of an object of the present invention is preparing the application in medical instrument, the medical treatment
Instrument is for preventing Asherman's syndrom or other position adhesions and tissue repair.
As the mode that further carries out, the medical instrument is three-dimensional porous rack；Either, the medical instrument is
Medicine three-dimensional porous rack is carried, the medicine three-dimensional porous rack that carries includes the three-dimensional porous rack, the three-dimensional porous rack
Load has drug；Either, the medical instrument is that BMSCs cell co-cultures three-dimensional porous rack, and the BMSCs cell is trained altogether
Feeding three-dimensional porous rack includes the three-dimensional porous rack, and three-dimensional porous rack and BMSCs cell are co-cultured 1-2 weeks, to
BMSCs cell is sufficiently proliferated in the hole of three-dimensional porous rack, grows, and it is three-dimensional porous to can be obtained the co-cultivation of BMSCs cell
Bracket.
It is the present invention preferably specific embodiment below, used raw material, equipment etc. are except spy in the following embodiments
Different restriction is outer can be obtained by buying pattern.It should be understood that these embodiments are to be not limited to limit for illustrating the present invention
The scope of the present invention.Implementation condition used in the examples can do further adjustment, the implementation being not specified according to actual conditions
Condition is usually conventional laboratory conditions.
Embodiment 1:
A kind of preparation method of three-dimensional porous rack, comprising:
1) size tunable the preparation step of monodisperse emulsion drop: is prepared with Nei phase using micro fluidic device, foreign minister
Monodisperse emulsion drop, monodisperse emulsion drop is cleaned and is collected；
Wherein, wherein foreign minister includes that degradable natural biologic material synthesizes high molecular material group with degradable artificial
At compound；The weight ratio that the degradable natural biologic material synthesizes high molecular material with degradable artificial is 1:
7.5.The degradable natural biologic material is sodium alginate.The artificial synthesized high molecular material is two propylene of polyethylene glycol
Acid esters.
The interior phase is methyl-silicone oil.
2) preparation step of emulsion droplet template: the monodisperse emulsion drop being collected into is stood, monodisperse emulsion drop
It can be self-assembled into close-packed structure, ultimately form uniform neat emulsion droplet template；Then emulsion droplet template is carried out
Curing process, the detailed process of curing process are as follows: one-step solidification is carried out to emulsion droplet template using ultraviolet light, when one-step solidification
Between be 2~5min, then emulsion droplet template is dipped in calcium chloride solution and carries out secondary curing, the secondary curing time be 2~
5min；
3) collection step of three-dimensional porous rack: collecting the emulsion droplet template after solidification and clean, cleaning it is specific
Process are as follows: the emulsion droplet template after solidification is immersed in methylene chloride, is impregnated twice, each 30min, by emulsion droplet template
In interior phase completely remove, then replace removal methylene chloride with alcohol gradient, obtain that hole is uniform, is mutually communicated, distribution rule
Three-dimensional porous rack.
Through detecting, the aperture for the three-dimensional porous rack that the present embodiment is prepared is 255~265 μm.
Embodiment 2:
1) size tunable the preparation step of monodisperse emulsion drop: is prepared with Nei phase using micro fluidic device, foreign minister
Monodisperse emulsion drop, monodisperse emulsion drop is cleaned and is collected；
Wherein, 1 foreign minister includes that degradable natural biologic material synthesizes what high molecular material formed with degradable artificial
Compound；The weight ratio of the degradable natural biologic material and degradable synthesising biological material is 1:7.5.It is described to drop
The natural biologic material of solution is sodium alginate.The synthesising biological material is methacrylic anhydride gelatin (Gelma).
The interior phase is methyl-silicone oil.
2) preparation step of emulsion droplet template: the monodisperse emulsion drop being collected into is stood, monodisperse emulsion drop
It can be self-assembled into close-packed structure, ultimately form uniform neat emulsion droplet template；Then emulsion droplet template is carried out
Curing process, the detailed process of curing process are as follows: one-step solidification is carried out to emulsion droplet template using ultraviolet light, when one-step solidification
Between be 2~5min, then emulsion droplet template is dipped in calcium chloride solution and carries out secondary curing, the secondary curing time 2~
5min；
3) collection step of three-dimensional porous rack: collecting the emulsion droplet template after solidification and clean, cleaning it is specific
Process are as follows: the emulsion droplet template after solidification is immersed in dehydrated alcohol, ultrasound 15s in Ultrasound Instrument is placed in, is changed after taking-up
It is ultrasonic again to enter new dehydrated alcohol, three times, the interior phase in emulsion droplet template is completely removed for so cleaning；Then by material
It impregnates in deionized water, displaces extra dehydrated alcohol, after dry, obtain that hole is uniform, be mutually communicated, distribution rule
Three-dimensional porous rack.
Referring to Fig. 2, wherein shown in Fig. 2 C and Fig. 2 D: the three-dimensional porous rack tool that the present embodiment is prepared
The characteristics of hole is uniform, be mutually communicated, distribution rule.
Through detecting, the aperture for the three-dimensional porous rack that the present embodiment is prepared is 255~265 μm.
Embodiment 3:
A kind of preparation method carrying medicine three-dimensional porous rack: three-dimensional porous rack described in embodiment 3 is soaked in 75% wine
In smart solution overnight, it then is cleaned repeatedly with sterile PBS (PH=7.4), and be immersed in ultraviolet irradiation 3h in PBS.By the porous branch
Frame is dipped in the bFGF solution of 100ug/mL, keeps drug fully dispersed into hole, takes out after 24 hours spare, can be obtained load
Medicine three-dimensional porous rack.
Carry application of the medicine three-dimensional porous rack in Asherman's syndrom rat model:
(1) foundation of Asherman's syndrom rat model: rat 350mg/kg10% chloral hydrate anesthesia shaves lower abdomen
Dorsal position is fixed on surgical plate after hair, iodophor disinfection, a stringer notch for being about 2 ~ 3cm is done in lower abdomen center, into abdomen
Chamber exposes uterus.After slowly choosing Y-shaped uterus, right uterine is taken, longitudinally slit 1.5 centimetres of notch from uterus top 1/3,
Fixed uterus is clamped with ophthalmology, blade scrapes endometrial tissue until inner membrance is congested, and gland mouth is impaired, and uterine wall is belittled semi-transparent
It is bright to stop operation.Isotonic saline solution rinses uterus to prevent inner membrance residual.
(2) it carries the verifying of medicine three-dimensional porous rack in a model: the three-dimensional porous rack for carrying bFGF is placed in uterine cavity
Injury region, with 6-0 absorbable suture interrupted suture.After rinsing abdominal cavity with salt water, with discontinuous 4-0 silk suture rectus
Fascia and skin.
Postoperative 1 week and 6 weeks Histological assessments show, compared with spontaneous healing/porous support group, carry the porous support of bFGF
Group endometrium and muscle layer are thicker, and arrangement of collagen fibers shows more orderly, and cicatrization is less, there is a large amount of blood vessels, gland
Body and cell；Referring to Fig. 3-4, as a result prompt: the porous support for carrying bFGF can prevent Asherman's syndrom, rush after rat uterus damage
Into the reparation of inner membrance, tissue vascularization and cellularised is improved, is hopeful to improve uterogestation functional rehabilitation.
Embodiment 4:
A kind of preparation method carrying medicine three-dimensional porous rack: medicine three-dimensional porous rack bubble will be carried in the FITC- of 20ug/ml
In BSA solution, keep drug fully dispersed into hole, taken out after standing overnight, can be obtained and carry medicine three-dimensional porous rack.
Detecting it and uploading rate is 30%, and the Cumulative release amount for monitoring week age can reach 40%.
Embodiment 5:
A kind of BMSCs cell co-cultivation three-dimensional porous rack, including three-dimensional porous rack as described in example 2, will be three-dimensional
Porous support and BMSCs cell co-culture 1-2 weeks, are sufficiently proliferated, give birth in the hole of three-dimensional porous rack to BMSCs cell
It is long, it can be obtained BMSCs cell and co-culture three-dimensional porous rack.
The above embodiment is only the preferred embodiment of the present invention, and the scope of protection of the present invention is not limited thereto,
The variation and replacement for any unsubstantiality that those skilled in the art is done on the basis of the present invention belong to institute of the present invention
Claimed range.
Claims (6)
1. a kind of three-dimensional porous rack, which is characterized in that the three-dimensional porous rack is degradable natural biologic material and can
The composite biological material of the artificial synthesized high molecular material composition of degradation；The three-dimensional porous rack has hole uniform, mutual
It penetrates through, the three-dimensional porous structure of distribution rule, aperture is 255~265 μm;The degradable natural biologic material with can
The weight ratio of the artificial synthesized high molecular material of degradation is 1~4:1~20;The degradable natural biologic material is seaweed
The one or more of sour sodium, collagen, chitosan, fibroin albumen；The artificial synthesized high molecular material is methacrylic acid
The one or more of acid anhydride gelatin, polyethyleneglycol diacrylate;
The preparation method of the three-dimensional porous rack, comprising:
The preparation step of monodisperse emulsion drop: single point of size tunable is prepared using micro fluidic device, foreign minister and Nei phase
Emulsion droplet is dissipated, monodisperse emulsion drop is cleaned and collected；Wherein, foreign minister is composite biological material；Foreign minister and Nei phase
It is immiscible；The flow velocity of foreign minister is 1~5mL/h；The flow velocity of interior phase is 0.1~0.3mL/h；The preparation step of emulsion droplet template
In, the detailed process of curing process are as follows: one-step solidification is carried out to emulsion droplet template using ultraviolet light, the one-step solidification time is 2
~5min, then emulsion droplet template is dipped in calcium chloride solution or cool down carry out secondary curing, the secondary curing time 2~
5min;
The preparation step of emulsion droplet template: the monodisperse emulsion drop being collected into is stood, or by monodisperse emulsion liquid
Drop is collected into and stands by using in purpose and the previously prepared mold of various shapes of demand, and monodisperse emulsion drop can self assembly
At close-packed structure, neat uniform emulsion droplet template is ultimately formed, emulsion droplet template is then subjected to curing process；
The collection step of three-dimensional porous rack: collecting the emulsion droplet template after solidification and clean, removes emulsion droplet template
In interior phase, obtain that hole is uniform, is mutually communicated, the three-dimensional porous rack of distribution rule.
2. three-dimensional porous rack as described in claim 1, which is characterized in that the three-dimensional porous rack further includes light-initiated
Agent, polyethylene glycol propylene glycol-polyethylene glycol, lauryl sodium sulfate；Photoinitiator, polyethylene glycol propylene glycol-poly- second
The weight ratio of glycol, lauryl sodium sulfate and composite biological material is 1:2:2:10~50.
3. three-dimensional porous rack as described in claim 1, which is characterized in that the three-dimensional porous rack can be by using purpose
Different shape and size are prepared into demand.
4. a kind of load medicine three-dimensional porous rack, which is characterized in that including as claimed in any one of claims 1-3 three-dimensional porous
Bracket, the three-dimensional porous rack load have drug.
5. a kind of marrow stromal cell co-cultures three-dimensional porous rack, which is characterized in that including such as claim 1-3 any one
Three-dimensional porous rack and marrow stromal cell are co-cultured 1~2 week, are existed to marrow stromal cell by the three-dimensional porous rack
It is sufficiently proliferated, grows in the hole of three-dimensional porous rack, can be obtained marrow stromal cell and co-culture three-dimensional porous rack.
6. a kind of three-dimensional porous rack as claimed in any one of claims 1-3 is preparing the application in medical instrument, special
Sign is that the medical instrument is for preventing Asherman's syndrom.
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| CN109897387A (en) * | 2019-01-25 | 2019-06-18 | 华南理工大学 | Application, porous gel and its preparation of a kind of modified gelatin in water packet air lotion |
| CN110834411B (en) * | 2019-11-18 | 2021-11-30 | 上海大学 | Cell-loaded three-dimensional bubble graphene-PEGDA-GelMA photocuring biological material, preparation method and application |
| CN113528424A (en) * | 2021-08-25 | 2021-10-22 | 中国人民解放军陆军特色医学中心 | Photosensitive biological material porous scaffold and application thereof |
| CN114470305B (en) * | 2022-03-09 | 2023-01-03 | 南方科技大学 | Hemostatic sponge and preparation method thereof |
| CN114854677B (en) * | 2022-07-04 | 2022-11-04 | 南京农业大学 | Microfluidic bionic fiber for cell culture meat production and preparation method and application thereof |
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