CN109970998A - A kind of method and application preparing GelMA super porous hydrogel with Pickering emulsion method - Google Patents
A kind of method and application preparing GelMA super porous hydrogel with Pickering emulsion method Download PDFInfo
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Abstract
The invention discloses a kind of method and application for preparing GelMA super porous hydrogel with Pickering emulsion method, GelMA and PEG-4SH are dissolved in PBS buffer solution by this method, stirring；After completely dissolution, MgO nano particle, stirring, ultrasound is added；Dodecane and photoinitiator is added, is vigorously stirred with high-shear homogenizer, obtains stable Pickering lotion；By lotion through UV illumination, plastic sufficiently washs dialysis through second alcohol and water, obtains GelMA super porous hydrogel.GelMA super porous hydrogel obtained has the advantage of macroporous structure, can promote the conveying of nutriment, the discharge of metabolite and intercellular exchange；And contain the ingredient of Mg element, have the function of promoting adherency, proliferation to Marrow Mesenchymal Stem Cells and promotes Osteoblast Differentiation, have excellent biocompatibility, can be used as the bone tissue engineering scaffold of function admirable, there is huge clinical application potential in Bone Defect Repari field.
Description
Technical field
The present invention relates to the technical fields of hydrogel material, refer in particular to a kind of with Pickering emulsion method preparation GelMA
The method and application of super porous hydrogel.
Background technique
Bone injury has been clinical common surgical disease, and used Osteoimplant on clinical treatment, mainly has certainly at present
Body bone, homogeneous allogenic bone and bone- xenograft, these three clinical Osteoimplants respectively have advantage and disadvantage, and limited source, therefore annual clinical
Treatment is required to a large amount of bone renovating material.Various bone renovating materials used in clinic also have certain problems, such as
Medical metal material osteoconductive is poor, mismatches with the elasticity modulus of human body natural bone tissue；Calcium phosphate bone cement is mechanical strong
It is poor to spend, it is impossible to be used in the reparation of load-bearing bone；Bioactivity glass degradation speed and bone growth speed mismatch, and lead to bone group
Knit the problems such as speed of growth is excessively slow or bone defect healing is imperfect.
Bone tissue complicated component contains lot of trace metallic element, such as Mg, Sr, Zn, Fe, Cu in the middle.In Bone Defect Repari material
In the research of material, the Bone Defect Repari performance of material is can be improved in incorporation minor metallic element.In lot of trace metallic element, Mg member
Element participates in the physiology courses such as bone tissue formation, bone metabolism and bone crystallizable mineral, to the adherency of osteoblast, proliferation and Osteoblast Differentiation
Behavior has facilitation.
Macromolecule hydrogel is a kind of is crosslinked by hydrophilic macromolecule and the three-dimensional network that is formed is combined jointly with hydrone
The material system of composition.The material for being usually used in bone repair hydrogel preparation has collagen, gelatin, fibroin albumen, chitosan, alginic acid
The natural biomass macromolecule such as sodium and hyaluronic acid.The hydrogel made of these natural polymers, on ingredient with body
Extracellular matrix is similar, while having good biology performance and degradation property, and cell is promoted to adhere on material etc.
Advantage is widely used in the research of Bone Defect Repari implantation material.There are many preparation methods by natural biomass high score at present
The hydrogel scaffold of variform is made in sub- material.
Large pore gel bracket organizational project, in terms of have a great application value, phase inside hydrogel
Intercommunicated hole configurations can promote the conveying of nutriment, the discharge of metabolite and intercellular exchange, can be used as
Good tissue engineering bracket.Hydrogel of the effective aperture in the μ m of 10nm~10 is generally known as microporous, is had
Hydrogel of the effect aperture greater than 10 μm is known as super porous hydrogel.It is commonly used for the method for preparing the macropore bracket of bio-medical at present
In or intercommunicating pore can not be formed or need to introduce extra chemicals increase toxicity, be unfavorable for the biocompatibility of material.
The design prepares the principle and feature of super porous hydrogel timbering material according to Pickering emulsion method, using MgO nanoparticle as
The stabilizer of Pickering lotion proposes and counts a kind of GelMA super porous hydrogel bracket, the bone repairing support as function admirable.
Summary of the invention
The purpose of the present invention is to overcome the shortcomings of the existing technology and deficiency, proposes one kind with Pickering emulsion method
The method and application of GelMA super porous hydrogel are prepared, the super porous hydrogel of this method preparation has the advantage of macroporous structure, can
Promote the conveying of nutriment, the discharge and intercellular exchange of metabolite；And the ingredient of Mg element is contained, to mouse
Mesenchymal stem cell has the function of promoting adherency, proliferation and promotes Osteoblast Differentiation, has excellent biocompatibility, can
As good tissue engineering bracket, there is huge clinical application potential in Bone Defect Repari field.
To achieve the above object, technical solution provided by the present invention are as follows: a kind of with the preparation of Pickering emulsion method
The method of GelMA super porous hydrogel obtains methyl-prop firstly, carrying out graft modification to gelatin chains with methacrylic anhydride
The gelatin of olefin(e) acid, i.e. GelMA, using double bond thereon, under the catalysis of photoinitiator I 2959, under UV illumination with four arms
PEG-4SH on sulfydryl occur thiol-ene click-reaction, hydrogel is quickly formed, by gel precursor liquid, i.e. water phase
It is middle that the oil-phase solution being made of MgO nano particle and dodecane is added, stable uniform is prepared under homogenizer high-speed stirred
Pickering lotion, then UV illumination plastic, wash away and are to get to MgO nano particle as the dodecane oil droplet of template
The GelMA super porous hydrogel of Pickering emulsion stabilizer.
It the described method comprises the following steps:
1) GelMA and PEG-4SH are dissolved in PBS (1 ×) buffer, obtain aqueous phase solution；
2) MgO nano particle is added in dodecane, re-ultrasonic dispersion after stirring obtains oil-phase solution；
3) water phase is mixed with oil-phase solution, 2959 photoinitiator of I is added, with homogenizer high-speed stirred, be made uniform steady
Fixed Pickering lotion；
4) lotion is injected in PDMS mold, through UV illumination, lotion plastic, demoulding is saturating with dehydrated alcohol and deionized water
Analysis is lyophilized to get taking MgO nano particle as the GelMA super porous hydrogel of Pickering emulsion stabilizer.
In step 1), molecular weight of the degree of substitution greater than 70%, PEG of the GelMA is 2~20kDa.
In step 1), the mass ratio of the GelMA and PEG-4SH are 1:0.5~1.
In step 2), the partial size of the MgO nano particle is 20~100nm.
In step 2), the time of the ultrasound is 10~60min.
In step 3), the additional amount of 2959 photoinitiator of I is 0.01~0.1wt%.
In step 3), the revolving speed of the homogenizer high-speed stirred is 1~20,000 turns/min, and mixing time is 30~90s.
In step 4), the PDMS mold with a thickness of 2~5mm, aperture is 5~10mm, and UV light application time is mold
Each 30~the 90s in positive and negative.
The GelMA super porous hydrogel as made from the above method is applied to Bone Defect Repari as tissue engineering bracket.
Compared with prior art, the present invention have the following advantages that with the utility model has the advantages that
The GelMA super porous hydrogel material that the present invention obtains has good biocompatibility and degradability.thiol-
Ene click-reaction mild condition, gelation time is short, and combines the cell adhesion of gelatin and the physicochemical property that PEG is excellent,
Prepare one kind on ingredient be it is uniform, cell compatibility is good, and is adjustable group in chemistry and mechanical properties
Engineering scaffold material is knitted, natural extracellular matrix is preferably simulated.The introducing of MgO nanoparticle, serves not only as Pickering
Emulsion stabilizer imparts material macroporous structure, promotes the conveying of nutriment, the discharge of metabolite and intercellular friendship
Stream, also effectively increases the mechanical strength of material, and Mg2+Release can effectively facilitate cell proliferation and Osteoblast Differentiation energy
Power is expected to become excellent bone repair hydrogel bracket.With huge clinical application potential.
Detailed description of the invention
Fig. 1 is the gelling mechanism figure of GelMA super porous hydrogel.
Fig. 2 is GelMA and gelatin1H NMR。
Fig. 3 is the contact angle test photo of MgO nanoparticle.
Fig. 4 is the compression modulus test chart of GelMA super porous hydrogel bracket.
Fig. 5 a is the EDS energy spectrum diagram of GelMA super porous hydrogel bracket (integral face is swept).
Fig. 5 b is the EDS energy spectrum diagram (partial sweep) of GelMA super porous hydrogel bracket.
Fig. 6 a is the Mg of GelMA super porous hydrogel bracket2+One of In-vitro release curves figure.
Fig. 6 b is the Mg of GelMA super porous hydrogel bracket2+The two of In-vitro release curves figure.
Fig. 7 a, Fig. 7 b, Fig. 7 c, Fig. 7 d be different MgO concentrations GelMA super porous hydrogel bracket SEM photograph.
Fig. 8 is that mBMSCs is planted on the GelMA super porous hydrogel bracket of different MgO concentrations, 1,3,7 days proliferation of cell
Situation map.
Fig. 9 is that mBMSCs is planted on the GelMA super porous hydrogel bracket of different MgO concentrations, and culture was by the 7th day, Live/
The laser co-focusing photo of Dead dyeing.
Specific embodiment
Specific implementation of the invention is described further below in conjunction with example, but embodiments of the present invention are not limited to
This.
Embodiment 1 (modification and characterization of gelatin)
One, the modification of gelatin
GelMA is to be obtained by gelatin through methacrylic anhydride modification, bright by the way that methacrylic anhydride to be grafted to
On xanthan molecule chain, the high carbon-carbon double bond of reactivity is introduced, in gelatin chains conducive on the PEG-4SH molecule with four arms
Thiol-ene click-reaction occurs for sulfydryl, is cross-linked into gel network under UV illumination.The gelling mechanism of GelMA super porous hydrogel
See Fig. 1.
5g gelatin is dissolved in 50mL PBS (1 ×) buffer, and in 60 DEG C of water-baths, stirring is to being completely dissolved.10mL methyl-prop
Olefin(e) acid acid anhydride instills the gelatin solution in stirring under the rate of 0.5mL/min, and 50 DEG C of water-baths are reacted 3 hours.It is slightly cooling, with 40
DEG C PBS (1 ×) diluting reaction object to 5 times, with terminate reaction.It is dialysed 7 days with the bag filter of 12-14kDa, in 40 DEG C of water-baths
Dialysis, is finally lyophilized, obtains product and store at 4 DEG C.
Two, the 1H NMR characterization of gelatin modified outcome
Nuclear magnetic resonance spectroscopy (1H NMR) analysis is carried out to above-mentioned gelatin and GelMA, measures its molecular structure, two kinds of molecules
1H H NMR spectroscopy see Fig. 2.
Spectral line is GelMA above Fig. 2, and lower section spectral line is gelatin, it can be seen that two nuclear-magnetism curves are almost the same, GelMA
There are two chemical shifts, the i.e. characteristic peak of methacryl amido at 5.35ppm and 5.6ppm, illustrate methacrylic anhydride
Success is modified onto gelatin main chain.
Embodiment 2 (contact angle test of MgO nano particle)
Pickering lotion is using ultrafine particle as emulsion obtained from emulsifier.Assuming that solids are
Spherical, state of the particle in grease phase, particle-water, particle-oil, the surface between water-oil can be for the internus of lotion
It is most important.If contact angle θ < 90 ° between particle and water phase, i.e. the wetability of expression solids and water phase is preferable, at this time
System easily forms oil-in-water (O/W) lotion；If the wetability of contact angle θ > 90 ° between particle and water phase, i.e. particle and oily phase
More preferably, system easily forms Water-In-Oil (W/O) lotion.So we can substantially judge lotion by the contact angle of particle and water
Type, and the hydrophilic and hydrophobic of particle must be moderate, too hydrophilic or too hydrophobic be all unable to get stable lotion.
MgO powder is dispersed in dehydrated alcohol, ultrasonic 30min, it is fully dispersed uniform, it then drips in very thin glass circle
On piece is volatilized completely to ethyl alcohol, then is dripped, and MgO powder is uniformly coated on glass wafer, film forming, then carries out contact angle
Test.The contact angle test photo of MgO nanoparticle is shown in Fig. 3.Measuring MgO nanoparticle contact angle test average value is 36.1 °.
It is measuring the result shows that, MgO nanoparticle is hydrophilic inorganic particle, can form stable O/W lotion.
Embodiment 3 (compression performance of GelMA super porous hydrogel bracket is tested)
0.05g GelMA is dissolved in 0.25mL PBS solution (1 ×), stirring to abundant dissolution.By 0.25mL 0.2g/
PEG-4SH (10,000 molecular weight) solution of mL is uniformly mixed with GelMA solution.0.5mL dodecane is added in 1.5mg nano-MgO
In, ultrasonic 15min.GelMA, PEG-4SH, dodecane, 2959 photoinitiator of I are mixed, with homogenizer in 1.6 ten thousand turns/min
Lower stirring 60s.Lotion is transferred to thick 5mm, in the PDMS mold of diameter 1cm, UV illumination, each 60s in positive and negative.It is final to be made
GelMA super porous hydrogel bracket for compression modulus test.The pressure of 0.3wt% and the hydrogel scaffold of 0.5wt%MgO content
Contracting curve is shown in Fig. 4.
The group content of MgO of 0.5wt% is higher, and emulsion droplet size is smaller, therefore the aperture of hydrogel is smaller, so content of MgO
High sample, compression modulus is higher, meets universal law.By the slope of 20% deformation region preceding in figure, calculate, 0.3wt%
The sample of content of MgO, compression modulus 6.99kPa；The sample of 0.5wt%MgO content, compression modulus 12.33kPa.
Embodiment 4 (elemental analysis of GelMA super porous hydrogel bracket)
0.1g GelMA is dissolved in 0.25mL PBS solution (1 ×), stirring to abundant dissolution.By 0.25mL 0.2g/mL
PEG-4SH (8,000 molecular weight) solution be uniformly mixed with GelMA solution.2.5mg nano-MgO is added in 0.5mL dodecane,
Ultrasonic 20min.GelMA, PEG-4SH, dodecane, 2959 photoinitiator of I are mixed, stirred at 20,000 turns/min with homogenizer
60s.Lotion is transferred to thick 2mm, in the PDMS mold of diameter 8mm, UV illumination, each 90s in positive and negative.Demoulding, through dehydrated alcohol
After deionized water dialysis 7 days, the pre-freeze under the conditions of -20 DEG C, then be lyophilized, it is finally made for constituent content qualitative analysis
GelMA super porous hydrogel bracket.
By the bracket of freeze-drying with liquid nitrogen quench forging after break into two with one's hands, be fixed on Electronic Speculum platform with conducting resinl, metal spraying 60s, with scanning electricity
Sub- microscope carries out the test of EDS power spectrum, the constituent content of qualitative analysis GelMA super porous hydrogel bracket.The entirety of EDS energy spectrum diagram
Face sweeps result and sees Fig. 5 a, and partial sweep result is shown in Fig. 5 b.It selects and is scanned around some hole, the border circular areas indicated in figure
Interior, Mg constituent content is 1.14wt%, higher than the content of Mg element within the scope of holistic vision.
(the Mg of GelMA super porous hydrogel bracket of embodiment 52+Release in vitro quantitatively characterizing)
The each group hydrogel scaffold of freeze-drying is weighed, immerses the concentration in 1ml PBS (pH=7.4) according to 10mg bracket,
It is placed a stent into 24 orifice plates respectively, the PBS solution that corresponding amount is added is impregnated.It incubates 14 days and is obtained often in 37 DEG C of constant-temperature tables
The magnesium ion release profiles of group.Shaking speed is 100rpm.In each regular time point, PBS solution is taken out, inductance is passed through
Coupled plasma optical emission spectroscopic methodology (ICP-OES, Perkin Elmer, Optima 2100DV, USA) detection magnesium ion is released
It puts.The PBS solution more renewed daily.The Mg of GelMA super porous hydrogel bracket2+In-vitro release curves are shown in Fig. 6 a and Fig. 6 b.
Fig. 6 a is daily Mg Element release amount, has added the group of MgO, Mg2+Release, have a burst release process, it is preceding
Two days contents are very high, and third day starts, and content reduces, and after 7 days, burst size tends towards stability, and concentration is lower.First 5 days, daily
Burst size within the scope of 20-500ppm.Fig. 6 b is that the Cumulative release amount of Mg element in 10 days tends towards stability after 7 days.
Embodiment 6 (internal morphology of the GelMA super porous hydrogel bracket of different MgO concentrations characterizes)
By each group hydrogel scaffold of freeze-drying with liquid nitrogen quench forging after break into two with one's hands, be fixed on Electronic Speculum platform with conducting resinl, metal spraying
60s observes the section of each group hydrogel scaffold, with scanning electron microscope to observe internal morphology and the aperture of hydrogel scaffold
Rule.The SEM photograph of the GelMA super porous hydrogel bracket of different MgO concentrations is shown in Fig. 7.(a:0wt%MgO；B:0.3wt%MgO；
C:0.5wt%MgO；D:0.7wt%MgO) without the group of Pickering emulsion method pore-creating, hydrogel does not have uniform pore structure,
Due to mechanical strength fragility, same processing method, this group of shrinkage of gel internal morphology, collapsing situation are serious, and hole wall is in scale
Shape.
0.3wt%MgO group: there is uniform macroporous structure, aperture is at 180 μm or so.
0.5wt%MgO group: there is uniform macroporous structure, aperture is at 110 μm or so.
0.7wt%MgO group: the uniformity in hole is weaker, and aperture is at 90 μm or so.
Aperture meets theoretically, and content of MgO is lower, the bigger trend in aperture, and pore structure is also more uniform.
Embodiment 7 (cell compatibility of the GelMA super porous hydrogel bracket of different MgO concentrations is tested)
Will be by thick 2mm, the hydrogel scaffold prepared in the PDMS mold of diameter 8mm is dialysed, it is lyophilized, after sterilizing, kind
The mBMSCs in upper 9th generation was planted, planting density is 50,000 cells/bracket.Change a not good liquor within every two days.
CCK-8 detection was carried out to culture to 1,3,7 day bracket, the absorbance of CCK-8 working solution is measured with microplate reader, with
Proliferative conditions of the quantitative analysis cell on bracket.1,3,7 days proliferative conditions of cell are shown in Fig. 8.
As seen from Figure 8, when culture was by the 3rd day, the only group of 0.5wt% is slightly proliferated, and first three groups are all not
Proliferation.When culture was by the 7th day, the nonporous hydrogel group of 0wt% is not proliferated yet, the group of 0.1wt%, 0.3wt%
It is slightly proliferated, the group proliferation of 0.5wt% is obvious.Illustrate, is macropore compared to nonporous gel when MgO concentration is lower
Structural advantage promotes cell Proliferation, but effect is unobvious；When MgO concentration is higher, although aperture reduces, Mg element promotees
Effect into cell Proliferation more highlights.
Live/Dead dyeing was carried out to culture to the 7th day bracket, with confocal laser scanning microscope cell in bracket
On distribution situation and cell state.7th day, the laser co-focusing photo of Live/Dead dyeing was shown in Fig. 9.
Illustrating the hydrogel scaffold with macroporous structure, proliferative conditions of the cell on bracket are more preferable, and cell quantity is more,
And cell state is preferable, in the state for sprawling attaching.When cell is grown, deeper region inside gel can be penetrated into, without
It is only to be grown in rack surface.
Embodiment described above is only the preferred embodiments of the invention, and but not intended to limit the scope of the present invention, therefore
All shapes according to the present invention change made by principle, should all be included within the scope of protection of the present invention.
Claims (10)
1. a kind of method for preparing GelMA super porous hydrogel with Pickering emulsion method, which is characterized in that firstly, using methyl-prop
Olefin(e) acid acid anhydride carries out graft modification to gelatin chains, obtains methacrylated gelatin, i.e. GelMA, using double bond thereon,
Under the catalysis of photoinitiator I 2959, thiol-ene occurs with the sulfydryl on the PEG-4SH of four arms under UV illumination and clicks instead
It answers, quickly forms hydrogel, by gel precursor liquid, i.e., the oil being made of MgO nano particle and dodecane being added in water phase
Phase solution prepares the Pickering lotion of stable uniform, then UV illumination plastic under homogenizer high-speed stirred, washes away as mould
The dodecane oil droplet of plate is to get the GelMA super porous hydrogel arrived with MgO nano particle for Pickering emulsion stabilizer.
2. a kind of method for preparing GelMA super porous hydrogel with Pickering emulsion method according to claim 1, special
Sign is, comprising the following steps:
1) GelMA and PEG-4SH are dissolved in PBS (1 ×) buffer, obtain aqueous phase solution；
2) MgO nano particle is added in dodecane, re-ultrasonic dispersion after stirring obtains oil-phase solution；
3) water phase is mixed with oil-phase solution, 2959 photoinitiator of I is added, with homogenizer high-speed stirred, stable homogeneous is made
Pickering lotion；
4) lotion is injected in PDMS mold, through UV illumination, lotion plastic, demoulding is frozen with dehydrated alcohol and deionized water dialysis
The dry GelMA super porous hydrogel to get with MgO nano particle for Pickering emulsion stabilizer.
3. a kind of method for preparing GelMA super porous hydrogel with Pickering emulsion method according to claim 2, special
Sign is: in step 1), molecular weight of the degree of substitution greater than 70%, PEG of the GelMA is 2~20kDa.
4. a kind of method for preparing GelMA super porous hydrogel with Pickering emulsion method according to claim 2, special
Sign is: in step 1), the mass ratio of the GelMA and PEG-4SH are 1:0.5~1.
5. a kind of method for preparing GelMA super porous hydrogel with Pickering emulsion method according to claim 2, special
Sign is: in step 2), the partial size of the MgO nano particle is 20~100nm.
6. a kind of method for preparing GelMA super porous hydrogel with Pickering emulsion method according to claim 2, special
Sign is: in step 2), the time of the ultrasound is 10~60min.
7. a kind of method for preparing GelMA super porous hydrogel with Pickering emulsion method according to claim 2, special
Sign is: in step 3), the additional amount of 2959 photoinitiator of I is 0.01~0.1wt%.
8. a kind of method for preparing GelMA super porous hydrogel with Pickering emulsion method according to claim 2, special
Sign is: in step 3), the revolving speed of the homogenizer high-speed stirred is 1~20,000 turns/min, and mixing time is 30~90s.
9. a kind of method for preparing GelMA super porous hydrogel with Pickering emulsion method according to claim 2, special
Sign is: in step 4), the PDMS mold with a thickness of 2~5mm, aperture is 5~10mm, and UV light application time is mold
Each 30~the 90s in positive and negative.
10. a kind of described in any item methods for preparing GelMA super porous hydrogel with Pickering emulsion method of claim 1 to 9
The application of GelMA super porous hydrogel obtained, it is characterised in that: the GelMA super porous hydrogel is answered as tissue engineering bracket
For Bone Defect Repari.
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