CN107551320A - A kind of 3D printing hydrogel porous support for possessing antibacterial functions and preparation method thereof - Google Patents
A kind of 3D printing hydrogel porous support for possessing antibacterial functions and preparation method thereof Download PDFInfo
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- CN107551320A CN107551320A CN201710610566.XA CN201710610566A CN107551320A CN 107551320 A CN107551320 A CN 107551320A CN 201710610566 A CN201710610566 A CN 201710610566A CN 107551320 A CN107551320 A CN 107551320A
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Abstract
The invention discloses a kind of 3D printing hydrogel porous support for possessing antibacterial functions and preparation method thereof, rack body is prepared for by 3D printing technique, the rack body is to be deposited to form layer by layer by the porous cubic unit of aquagel fibre structure, the surface modification of the rack body has antibacterial polypeptide layer, the component of the antibacterial polypeptide layer has liquaemin and poly-D-lysine, for improving the bioactivity of rack body, realize that antibacterial carries the function of medicine.Preparation process of the present invention is simple, assign the uniform hole of hydrogel and good antibacterial effect, improve its bioactivity simultaneously, reach pre- aseptic purpose after tissue repair, it is expected to be widely applied in tissue repair and reconstruction engineering, such as repair of cartilage, revascularization, skull reparation etc..
Description
Technical field
The present invention relates to tissue repair and the technical field rebuild, a kind of 3D printing water for possessing antibacterial functions is referred in particular to
Gel porous support and preparation method thereof.
Background technology
Tissue repair and the focus that in process of reconstruction, timbering material is always studied.Hydrogel is as a kind of soft with human body
Tissue has similar water content, biological micro-structural, good biocompatibility and mechanical property, is widely used in tissue and repaiies
In journey of returning to work.But gradually found in clinical practice, because the individual sex differernce of patient's defect is, it is necessary to during longer operation
Between form fit is carried out to Traditional bulk hydrogel implant and tissue defect, extend the operating time of patient, increase patient
Pain.Moreover, traditional block hydrogel because inside can not provide nutrition and oxygen, causes the necrosis of interior tissue material tight
Weight.Secondly, bacterial wound infection easily occurs for the physiological environment of hydrogel implant site uniqueness high humility, causes wound needs more
Recover for a long time.These all bring huge challenge to the clinical tissue engineer applied of hydrogel.
3D printing is a kind of based on CAD software aided design models, the rapid shaping technique successively accumulated.Because it has
Accurate quickly shaping manufacture advantage is widely used in field of tissue engineering technology.The hydrogel prepared by 3D printing technique has
Fine internal structure, complicated appearance and size, the internal void of stable homogeneous.What is more important its can according to patient from
The defect shape of body, with reference to computer display technology (Micro-CT etc.), personalized fast custom matching patient's defect pattern
Hydrogel porous support.But relative to the solid hydrogel of Traditional bulk, because porous scaffold surface product and internal void show
Write increase, humidity increase so that it is easier that postoperative bacterium infection occurs.Therefore, beating for hydrogel porous support how is promoted
Printing stability, hole stable homogeneous and prevention of postoperative infection turns at present by hydrogel applied to two weights in clinical tissue engineering
Want problem.
At present, it is that hydrogel substrate material is carried out into chemical graft that hydrogel material, which carries out the one of method of antimicrobial treatment,
Upper antimicrobial molecule, such as antibacterial polypeptide.But this method preparation process is grown, chemical reaction grafting rate is limited, so antibacterial effect
Unobvious.It is by physical absorption antibacterial metal ions, so that support has antibacterial effect to have method in addition.But this method
In the presence of certain limitation, Metal ion release is too fast and the bacteriostasis of metal ion is limited.
The content of the invention
It is an object of the invention to overcome the deficiencies in the prior art and shortcoming, there is provided a kind of 3D for possessing antibacterial functions is beaten
Hydrogel porous support and preparation method thereof is printed, preparation process is simple, assigns the uniform hole of hydrogel and good antibacterial effect
Fruit, while its bioactivity is improved, reach pre- aseptic purpose after tissue repair, be expected to be widely applied and repaiied for tissue
In multiple and reconstruction engineering, such as repair of cartilage, revascularization, skull reparation etc..
To achieve the above object, technical scheme provided by the present invention is as follows：
A kind of 3D printing hydrogel porous support for possessing antibacterial functions, including the rack body by 3D printing, it is described
Rack body is deposited formed layer by layer by the porous cubic unit of aquagel fibre structure, and the surface modification of the rack body has anti-
Bacterium layer polypeptide, the component of the antibacterial polypeptide layer have liquaemin and poly-D-lysine, real for improving the bioactivity of rack body
Existing antibacterial carries the function of medicine.
The liquaemin is 185USP units/mg, and the poly-D-lysine molecular weight is 2000-5000Da.
It is by through the modified gelatin of methacrylic anhydride that the rack body, which uses ink during 3D printing,
(GelMA), Sodium Hyaluronate (HAMe) and light trigger (I2959) are formulated, and configuration proportion is gelatin (GelMA)：Thoroughly
Bright matter acid sodium (HAMe)：Light trigger (I2959)=(20~30)：(3~5)：(0.1~1)；Wherein, the gelatin (GelMA)
Grafting rate be 80%~90%, the grafting rate of the Sodium Hyaluronate (HAMe) is 20%~30%, the light trigger
(I2959) it is 2- hydroxyls -4- (2- hydroxy ethoxies) -2- methyl phenyl ketones.
The rack body is the Light lnduced Free Radicals copolymer of gelatin (GelMA) and Sodium Hyaluronate (HAMe), and it contains
Water is up to more than 80%, and profile is porous tetrahedron, and hole is uniform.
A kind of preparation method for the 3D printing hydrogel porous support for possessing antibacterial functions, comprises the following steps：
1) chemical modification is carried out to gelatin (GelMA) and Sodium Hyaluronate (HAMe) material using methacrylic anhydride, made
It can polymerize under ultraviolet irradiation condition；
2) gelatin (GelMA) after chemical modification and Sodium Hyaluronate (HAMe) are configured into required 3D in proportion to beat
Printing ink, and add light trigger (I2959)；
3) hydrogel porous support threedimensional model is established by microcomputer modelling software, imports delamination software and be layered；
4) print parameters are adjusted, mixed solution is printed into hydrogel porous support by designing a model, it is low at≤20 DEG C
The lower property that is molded, can be solidified at low temperature by hydrogen bond using gelatin for the first time of temperature；Then the one-step forming of ultraviolet lighting second,
Trigger polymerizing curable using ultraviolet lighting；
5) LBL self-assembly (LBL) is carried out in hydrogel porous scaffold surface：First by the hydrogel porous support of preparation
After carrying out PBS, it is soaked in poly-D-lysine (PLL) solution 20~30 minutes, then takes out hydrogel porous support,
Surface is rinsed with PBS, then hydrogel porous support was soaked in heparin sodium aqua after 20~30 minutes again and taken out, PBS punchings
Surface is washed, is then repeatedly soaking in poly-D-lysine (PLL) solution 20~30 minutes, is repeated the above steps 5-7 times, last PBS washings
After be put in 4 DEG C of refrigerators and preserve.
Chemically modified gelatin is prepared with the following method：First by Gelatin in 40~50 DEG C of PBS solution,
Methacrylic anhydride is added dropwise, after being then stirred vigorously 1~4h of reaction, adds 45 DEG C of PBS solution stopped reaction, finally
Mixed solution is loaded into bag filter, dialysed 3-5 days in 40 DEG C of water, spongiform gelatin materials are obtained after lyophilized.
Chemically modified Sodium Hyaluronate is prepared with the following method：Sodium Hyaluronate is dissolved in 50 DEG C first
In PBS solution, methacrylic anhydride is then added dropwise, using 5M NaOH solution adjust reaction system to PH=8~9 it
Between, stirring reaction is stayed overnight at 50 DEG C, after reaction terminates, mixed liquor is poured into ethanol and recrystallizes Sodium Hyaluronate product
Come, filtering, be re-dissolved in deionized water, load bag filter, dialyse 3~5 days at room temperature, it is cotton-shaped to obtain sponge after lyophilized
Sodium Hyaluronate material.
The configuration concentration ratio of the 3D printing ink is：Gelatin concentration is 10%~15%, hyaluronic acid na concn model
Enclose for 1.5%~2.5%, photoinitiator concentration scope is 0.1%~0.5%.
3D printing major parameter is arranged to：It is 0~5 DEG C to print low temperature platform temperature, shower nozzle extrusion pressure is 0.8~
1.2bar, i.e. 8 × 104~1.2 × 105Pa, printing syringe needle XY translational speeds are 20~30mm/s；Ultraviolet ray intensity used is 5
~20mW/cm2, light application time are 5~20 minutes.
In step 5), mainly using 3D printing hydrogel porous surface it is made up of hyaluronic acid, is had in its structure
More carboxyl, there can be electrostatic reciprocation with cationic high-molecular-poly-D-lysine, form stable physics
Key.The concentration of the Poly-L-Lysine Solution is 2mg/mL, and the concentration of the heparin sodium aqua is 10mg/mL.
The present invention compared with prior art, has the following advantages that and beneficial effect：
1st, the present invention is innovative using hydrogel porous support of the 3D printing technique structure with good biocompatibility
Hydrogel rapid shaping is made using two-step method, the stable printing initial configuration for maintaining hydrogel, prevents internal void from caving in.Water
Gel porous support has fine internal structure, the internal void of stable homogeneous.What is more important its can be according to patient
The defect shape of itself, personalized fast custom matching patient's hydrogel recovery support, reduces patient suffering, accelerates to be cured
Close.
2nd, the present invention using LBL technical methods structure antibacterial polypeptide finishing coat, according to heparin, poly-D-lysine with
There is stronger electrostatic reciprocation principle in hydrogel surface, method is simple, easy to operate, be in hydrogel material surface modification
The effective antibacterial decorative layer of one layer of stabilization.Hydrogel porous support antibiotic layer slowly discharges PLL as hydrogel is slowly degraded,
Bacterial reproduction and growth are effectively prevented, to reach the purpose that prevention organizational project repairs postoperative infection, it is more to improve hydrogel
The application prospect of hole support.
Brief description of the drawings
Fig. 1 is the print procedure figure of hydrogel porous support.
Fig. 2 is the shape graph after 3D hydrogels porous support freezes.
Fig. 3 is that hydrogel porous support observes rack surface figure after the amplification of 3D microscopes.
Fig. 4 a be after the antibacterial polypeptide modification that comes to the surface hydrogel porous support to the proliferative effect of Escherichia coli and anti-
Bacterium can try hard to.
Fig. 4 b be after the antibacterial polypeptide modification that comes to the surface hydrogel porous support to the propagation shadow of staphylococcus aureus
Ring and antibacterial ability figure.
Embodiment
With reference to specific embodiment, the invention will be further described.
What the present embodiment was provided possesses the 3D printing hydrogel porous support of antibacterial functions, including the branch for passing through 3D printing
Frame body, the rack body are to be deposited to form layer by layer by the porous cubic unit of aquagel fibre structure, the rack body
Surface modification has antibacterial polypeptide layer, and the component of the antibacterial polypeptide layer has liquaemin and poly-D-lysine, for improving rack body
Bioactivity, realize antibacterial carry medicine function；Wherein, the liquaemin is 185USP units/mg, the poly-D-lysine
Molecular weight is 2000-5000Da.
It is by through the modified gelatin of methacrylic anhydride that the rack body, which uses ink during 3D printing,
(GelMA), Sodium Hyaluronate (HAMe) and light trigger (I2959) are formulated, and configuration proportion is gelatin (GelMA)：Thoroughly
Bright matter acid sodium (HAMe)：Light trigger (I2959)=(20~30)：(3~5)：(0.1~1)；Wherein, the gelatin (GelMA)
Grafting rate be 80%~90%, the grafting rate of the Sodium Hyaluronate (HAMe) is 20%~30%, the light trigger
(I2959) it is 2- hydroxyls -4- (2- hydroxy ethoxies) -2- methyl phenyl ketones.In a word, the rack body be gelatin (GelMA) with
The Light lnduced Free Radicals copolymer of Sodium Hyaluronate (HAMe), its water content are up to more than 80%, and profile is porous tetrahedron, hole
Gap is uniform.
It is the preparation method of the above-mentioned 3D printing hydrogel porous support of the present embodiment below, comprises the following steps：
1) 20g gelatin is dissolved in 200mL PBS solution, heating water bath to 50 DEG C of fully dissolvings.Using microsyringe
It is added dropwise 10mL methacrylic anhydrides, about 30 minutes.Rear keeping temperature is added dropwise and is stirred vigorously lower reaction 2h for 50 DEG C, adds
Enter the pure PBS solution dilution stopped reactions of 45 DEG C of 200mL, complete soln loading bag filter is dialysed.Start 2 days small every 4
When change a deionized water, respectively change a water sooner or later within latter 3 days.After dialysis, by solution carry out at -80 DEG C freeze obtain sponge
Shape methacrylic acid anhydridization gelatin (GelMA).
2) 3g Sodium Hyaluronates (HAMe) are dissolved in 50 DEG C of 300mL PBS solutions, 10mL first is then added dropwise
Base acrylic anhydride, between adjusting the PH to 8~9 of reaction system using 5M NaOH solution, stirring reaction is stayed overnight at 50 DEG C, instead
After should terminating, mixed liquor is poured into HAMe product Precipitations in 1000mL ethanol, filtering, is re-dissolved in deionized water
In, load bag filter, dialysed 5 days using the dialysis process of embodiment 1 at room temperature, it is lyophilized to obtain the cotton-shaped HAMe materials of sponge.
3) 3g GelMA are dissolved in 20mL PBS solutions at 50 DEG C successively, add 0.1g light trigger I2959, dissolving
After completely, 0.4g HAMe are added under the conditions of lucifuge dissolving are sufficiently stirred at 50 DEG C, ultrasound removes bubble in 10 minutes, you can
Marking ink used in 3D printing is obtained, is fitted into printing barrel, is stored in stand-by in 4 DEG C of refrigerators.
4) 3D printing model is built in computer, first cover half type is positive tetrahedron, long a width of 12mm, high 4mm, internal void
For 0.4mm × 0.4mm square openings.Printing syringe needle selects 400 μ m diameters.Then marking ink in 4 DEG C of refrigerators is taken out, be loaded into
3D-Bioprinter printing heads, it is that printing low temperature platform temperature is 4 DEG C to set print parameters, and shower nozzle extrusion pressure is
1.0bar, printing syringe needle XY translational speeds are 25mm/s.First one-step forming using GelMA can low temperature (≤20 DEG C) pass through hydrogen bond
The property of solidification, second step ultraviolet lighting trigger polymerizing curable, and ultraviolet ray intensity used is 20mW/cm2, and light application time is 5 points
Clock, obtain required hydrogel porous support.
5) the hydrogel porous support of 3D printing is used into LBL technologies, LBL self-assembly (LBL) mainly utilizes 3D printing
Hydrogel porous surface is made up of hyaluronic acid, there is more carboxyl, can be deposited with cation high molecular-poly-D-lysine
In electrostatic reciprocation, stable secondary or physical bond is formed.By hydrogel scaffold using first step immersion PLL first after PBS washings
Solution 20~30 minutes, PLL concentration are 2mg/mL, are washed 2 times using PBS.Second step immersion heparin sodium aqua 20~30 minutes,
Concentration is 10mg/mL, repeats self assembly 5 times, last layer is poly-D-lysine antibacterial polypeptide layer, you can obtains required having
The 3D printing hydrogel porous support of antibacterial functions.
Fig. 1, which is shown, is printed the 3D printing ink of preparation using low temperature 3D printer machine, is come from Fig. 2 and Fig. 3
See, solidify the hydrogel porous support of structure using two-step method, shape is regular, and internal void is uniform.3D is carried out to hydrogel to stand
The hydrogel scaffold of the micro- sem observation hygrometric state of body, can be clearly it was observed that the square opening that size is about 400 μm, pore size be equal
One.
Hydrogel porous support (the HAGelMA- after LBL technologies modify upper antibacterial polypeptide is shown in Fig. 4 a, 4b
HNP) to Escherichia coli negative bacterium (E.Coil) and S. aureus-positive bacterium (SA) antibacterial effect, from Fig. 4 a and Fig. 4 b's
As a result from the point of view of, and the antibacterial effect of the hydrogel porous support (HAGelMA) of non-surface modification and blank cultures (Control)
Contrast, the hydrogel scaffold by antibacterial polypeptide modification have the effective energy for killing bacterium and suppressing bacteriological aftergrowth breeding
Power, and unmodified porous aquagel bacterial multiplication is substantially vigorous.As a result show：The present invention is more using LBL technology modified antimicrobials
The 3D printing porous aquagel support of peptide possesses good fungistatic effect, has in regeneration and recovery project field wide
Application prospect, it is worthy to be popularized.
Examples of implementation described above are only the preferred embodiments of the invention, and the implementation model of the present invention is not limited with this
Enclose, therefore the change that all shape, principles according to the present invention are made, it all should cover within the scope of the present invention.
Claims (10)
1. a kind of 3D printing hydrogel porous support for possessing antibacterial functions, including the rack body by 3D printing, its feature
It is：The rack body is to be deposited to form layer by layer by the porous cubic unit of aquagel fibre structure, the table of the rack body
Face is modified with antibacterial polypeptide layer, and the component of the antibacterial polypeptide layer has liquaemin and poly-D-lysine, for improving rack body
Bioactivity, realize that antibacterial carries the function of medicine.
A kind of 2. 3D printing hydrogel porous support for possessing antibacterial functions according to claim 1, it is characterised in that：Institute
It is 185USP units/mg to state liquaemin, and the poly-D-lysine molecular weight is 2000-5000Da.
A kind of 3. 3D printing hydrogel porous support for possessing antibacterial functions according to claim 1, it is characterised in that：Institute
It is by through the modified gelatin of methacrylic anhydride, Sodium Hyaluronate and light to state rack body and use ink during 3D printing
Initiator is formulated, and configuration proportion is gelatin：Sodium Hyaluronate：Light trigger=(20~30)：(3~5)：(0.1~1)；
Wherein, the grafting rate of the gelatin is 80%~90%, and the grafting rate of the Sodium Hyaluronate is 20%~30%, and the light draws
Hair agent is 2- hydroxyls -4- (2- hydroxy ethoxies) -2- methyl phenyl ketones.
4. a kind of 3D printing hydrogel porous support for possessing antibacterial functions according to claim 1 or 3, its feature exist
In：The rack body is the Light lnduced Free Radicals copolymer of gelatin and Sodium Hyaluronate, and its water content is up to more than 80%, outside
Shape is porous tetrahedron, and hole is uniform.
A kind of 5. preparation method for the 3D printing hydrogel porous support for possessing antibacterial functions, it is characterised in that：Including following step
Suddenly：
1) chemical modification is carried out to gelatin and Sodium Hyaluronate material using methacrylic anhydride, can be in ultraviolet lighting bar
It polymerize under part；
2) gelatin after chemical modification and Sodium Hyaluronate are configured to required 3D printing ink in proportion, and adds light and draws
Send out agent；
3) hydrogel porous support threedimensional model is established by microcomputer modelling software, imports delamination software and be layered；
4) print parameters are adjusted, mixed solution are printed into hydrogel porous support by designing a model, in a low temperature of≤20 DEG C
It is molded for the first time, the property that can be solidified at low temperature by hydrogen bond using gelatin；Then the one-step forming of ultraviolet lighting second, utilize
Ultraviolet lighting triggers polymerizing curable；
5) LBL self-assembly is carried out in hydrogel porous scaffold surface：It is clear that the hydrogel porous support of preparation is subjected to PBS first
After washing, it is soaked in Poly-L-Lysine Solution 20~30 minutes, then takes out hydrogel porous support, surface is rinsed with PBS,
Then hydrogel porous support was soaked in heparin sodium aqua after 20~30 minutes again and taken out, PBS rinses surface, repeats to soak
In Poly-L-Lysine Solution 20~30 minutes, repeat the above steps 5~7 times, being put in 4 DEG C of refrigerators after last PBS washings preserves.
6. a kind of preparation method of 3D printing hydrogel porous support for possessing antibacterial functions according to claim 5, its
It is characterised by, chemically modified gelatin is prepared with the following method：First by PBS solution of the Gelatin at 40~50 DEG C
In, methacrylic anhydride is added dropwise, after being then stirred vigorously 1~4h of reaction, adds 40~50 DEG C of PBS solution and stops instead
Should, mixed solution is finally loaded into bag filter, dialysed 3~5 days in 40~50 DEG C of water, spongiform gelatin is obtained after lyophilized
Material.
7. a kind of preparation method of 3D printing hydrogel porous support for possessing antibacterial functions according to claim 5, its
It is characterised by：Chemically modified Sodium Hyaluronate is prepared with the following method：Sodium Hyaluronate is dissolved in 40~50 first
DEG C PBS solution in, methacrylic anhydride is then added dropwise, using 5M NaOH solution adjust reaction system to PH=8
Between~9, stirring reaction is stayed overnight at 40~50 DEG C, after reaction terminates, mixed liquor is poured into ethanol and produces Sodium Hyaluronate
Thing is recrystallized, and filtering, is re-dissolved in deionized water, loads bag filter, is dialysed 3~5 days at room temperature, is produced after lyophilized
The Sodium Hyaluronate material cotton-shaped to sponge.
8. a kind of preparation method of 3D printing hydrogel porous support for possessing antibacterial functions according to claim 5, its
It is characterised by, the configuration concentration ratio of the 3D printing ink is：Gelatin concentration is 10%~15%, hyaluronic acid na concn model
Enclose for 1.5%~2.5%, photoinitiator concentration scope is 0.1%~0.5%.
9. a kind of preparation method of 3D printing hydrogel porous support for possessing antibacterial functions according to claim 5, its
It is characterised by, 3D printing major parameter is arranged to：It is 0~5 DEG C to print low temperature platform temperature, shower nozzle extrusion pressure is 0.8~
1.2bar, i.e., 8 × 104~1.2 × 105Pa, printing syringe needle XY translational speeds are 20~30mm/s；Ultraviolet ray intensity used be 5~
20mW/cm2, light application time is 5~20 minutes.
10. a kind of preparation method of 3D printing hydrogel porous support for possessing antibacterial functions according to claim 5, its
It is characterised by：In step 5), the concentration of the Poly-L-Lysine Solution is 2mg/mL, and the concentration of the heparin sodium aqua is
10mg/mL。
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