CN104857574B - Intelligent nano-hydrogel topological structure antibacterial bone plate and its manufacture method - Google Patents
Intelligent nano-hydrogel topological structure antibacterial bone plate and its manufacture method Download PDFInfo
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- CN104857574B CN104857574B CN201510305277.XA CN201510305277A CN104857574B CN 104857574 B CN104857574 B CN 104857574B CN 201510305277 A CN201510305277 A CN 201510305277A CN 104857574 B CN104857574 B CN 104857574B
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Abstract
The invention discloses a kind of intelligent nano-hydrogel topological structure antibacterial bone plate, matrix surface covering is kernel by polyethylene glycol co acroleic acid microgels core, outer layer controlled release shell is pH responsive type polymethylacrylic acid microgel layer, both constitute intelligent charge gradient radial distribution and are combined protomere, the compound protomere coating is in stainless steel, titanium or titanium alloy bone plate form topological antibacterial structure surface and load beta-alexin -1~3, lactoferrin 1 11, LL 37, the similar body of histatins, protegrins 1 and artificial synthesized cationic antibacterial peptide.The present invention be allowed to play when bacterium is present in implants surface in the case that, its as stimulus " contact sterilization " intelligent stimuli responsive mechanism.So as to realize Gegenbaur's cell etc. in interior plant surface fast-growth and reduction bacterial adhesion and kill bacterium using antibacterial peptide, " target spot " of formation really suppression bacterial biof iotalm is caused to control in its initial period, finally to reduce the incidence of " implants infections relating ".
Description
Technical field
The invention belongs to surgical material and its manufacture method, specifically, intelligent nano-hydrogel topology knot
Structure antibacterial bone plate.
Background technology
According to international Internal Fixation Research Association AO (Arbeit sgem ein schaft fur Osteosyntheses
Fragen)/ASIF (Association for the Study of Internal Fixation) is reported, in open fracture
Infection rate is up to about 3~40%, the infection rate increase by more than 30% if using interior fixation.Every year in Yue200Wan Li institutes of the U.S.
In cases of infection, about 50% is relevant with implants, not only consumption up to 24,000,000,000 dollars of the huge wealth of society, also result in including
Bacterial resistance, the amputation even serious consequence such as annual 100000 death.To find out its cause, bacterium is adhered to shape with implant material
It is the main pathological basis of " implants infections relating " into complete biomembrane." implants infections relating " (Orthopedic
Device Related Infections, ODRI) it is to perplex one of most common and serious complication of Orthopedic Clinical all the time.
For ODRI, general traditional treatment method is：On the basis of implants are taken out, thorough debridement remove infection and necrosis bone with it is soft
Tissue and most of bacterium；Fracture site is fixed temporarily using exterior fixing rack；Whole body or the heavy dose of antibiotic of topical application disappear
Go out remaining bacteria；Wound is closed using technologies such as flaps.Really without row bone grafting, bone are moved or extended and film skeletonization skill again after infection
Art recovers bone structure.
For ODRI traditional treatment technology, cycle length, cost are big, use exterior fixing rack fix bone more, maintain it
Stability.And less use internal fixation device, with the implants such as open fracture and firearm injury light plate screw then by regarding
The reason for for contraindication, is identical, and only about quantity is 103CFU/ml bacterium can adhere to implants and and be formed completely
Biomembrane causes ODRI generation, development to be bases with continuing protracted course of disease.In the case of without implants, even if open
Fracture is local 106CFU/ml bacteriums, may not also cause human body to infect.But it is outer fixed have pin site infection rate it is high (5.9%~
19%), be not fixed easily in setting board of skeleton end fracture, draw point loosen and have a strong impact on minimal invasive treatment, be not easy to nursing the shortcomings of.And
In setting board of skeleton end fracture, bone plate is the mode of irreplaceable fixation fracture site.Therefore, in ODRI prevention and treatment,
Conventional antibiotic application method：Whole body or local application.The former, causes in whole body blood often due to local damage and blood follow destruction
Antibiotic is difficult to reach effective antimicrobial concentration in damage part；The latter, although the antibiosis of high concentration can be produced in local and initial
Element, but then rapid decrease, it is impossible to maintain effective treatment concentration.Therefore, it is difficult to the formation to ODRI and Traumatic osteomyelitis
Effective anti-infectious function is produced with development.Currently for bacterial biof iotalm, by suppressing the synthesis of its polysaccharide matrix and promoting it
Decomposition, interference density induction system plus with monoclonal antibody and the surface physicochemical property of implant is improved to reduce the viscous of bacterium and material
The methods such as attached and antibiotic coating, it was demonstrated that ODRI incidences can be reduced to a certain extent, but can not thoroughly solve life
The formation of thing film and thus caused persistent infection.Therefore, finding the method or thing of new change fixture materials surface characteristic
Matter as current clinical position in the urgent need to, be the multi-field research focus such as domestic and international microorganism, material, clinical medicine it
One.
The content of the invention
An object of the present invention is that providing a kind of anti-infective and suppression bacterial biof iotalm formation the intellectuality of strength receives
Rice hydrogel topological structure antibacterial bone plate, the bone plate prepares the intelligent charge gradient footpath that PEG-co-AA protomeres are core
To distribution gel particle, the compound protomere that pH responsive type PMMA microgels layer is controlled release shell.The protomere can not only be abundant
The antibacterial peptides such as hBD-3 are loaded, and due to the antibacterial peptide such as hBD-3 presentation positive charge gradient radial distribution in protomere core, are made
There is " intellectuality " automatically to be filled vacancies in the proper order from high potential to low potential, play " contact sterilization " effect and open up for the antibacterial peptides such as hBD-3
The nano-hydrogel coating of triple " intellectuality " features of antibacterial structure is flutterred, it is easy with the predicament and coating that overcome conventional crosslinking method
Hydrolysis, load few and half-life short the defect of antibacterial peptide amount, so as to break through the application that the antibacterial peptides such as hBD-3 prevent and treat ODRI in vivo
" bottleneck ".
The second object of the present invention is to provide a kind of manufacture of intelligent nano-hydrogel topological structure antibacterial bone plate
Method.
A kind of intelligent nano-hydrogel topological structure antibacterial bone plate, including titanium or titanium alloy bone plate (1), its
Key is：The topological antibacterial structure surface recombination protomere load beta-alexin -1 of titanium or the titanium alloy bone plate~
3rd, the similar body of lactoferrin 1-11, LL-37, histatins (histatin analogue DHVAR-5), endogenous antimicrobial
Polypeptide -1 (Protegrins-1) and artificial synthesized 0.025~1mg/mL of cationic antibacterial peptide per unit area concentration.The titanium
Metal or the covering of titanium alloy synthetism plate surface are by polyethylene glycol-co- acrylic acid (Poly (ethylene glycol)-co-
Acrylic acid, PEG-co-AA) microgel core be kernel, outer layer controlled release shell be pH responsive type polymethylacrylic acid (poly
The intelligent charge gradient radial distribution of (methacrylic acid, PMAA) microgel layer is combined protomere, and this is combined micro- glue
Grain coating forms topological antibacterial structure surface recombination protomere load beta-alexin 1 in stainless steel, titanium or titanium alloy bone plate
Element -1~3, lactoferrin 1-11, LL-37, the similar body of histatins (histatin analogue DHVAR-5), endogenous
Antimicrobial polypeptide -1 (Protegrins-1) and artificial synthesized cationic antibacterial peptide.The compound protomere diameter 100~
400 μm, 0.6~8 μm of altitude range.PEG-co-AA the microgels minus 6~40mv of core potential range, 1~1000nm of core internal diameter；
The PMAA microgels layer minus 6~0mv of potential range, 5~30 layers of the number of plies.The compound protomere possesses electric charge from high potential to low potential
Automatically the characteristic shifted.Between the topological antibacterial structure surface recombination protomere (3) of the stainless steel, titanium or titanium alloy bone plate
0.5~4 μm away from scope.
A kind of manufacture method of intelligent nano-hydrogel topological structure antibacterial bone plate, it is it is critical that by following step
It is rapid to carry out：
Step 1, titanium or (1) surface preparation of titanium alloy bone plate and titanium surface on-line treatment.Titanium or titanium are closed
Golden bone plate (1) is cleaned by ultrasonic with distilled water, acetone, 75% ethanol, distilled water successively, panel carries out hydrophilic treated
Each 10~20 minutes, insert 0.2mg/mL, mean molecule quantity is 30~50k, pH9 many PLLs (poly-l-lysine,
PLL) 2~3 hours, in titanium alloy substrate formation positively charged macromolecule layer, initial layer deposition is carried out, deionized water is used after taking-up
Clean, be dried at room temperature for stand-by.
Step 2, PEGDA/AA emulsions (2) are prepared.Make even respectively the polyethylene glycol acrylate of average molecular weight 500~600
(Polyethylene Glycol Diacrylate, PEGDA) 200 μ l, acrylic monomers (acrylic acid, AA) is at 50 DEG C
0~20 μ l, and the μ l of light trigger (Darocur 1173, Ciba) 10 are left and taken after distillation, co-dissolve is in dichloromethane
Oil phase is formed in (dichloromethane, DCM) 1ml.1~10% polyvinyl alcohol (PVA) 1~3ml is molten as emulsifying agent
Solution forms aqueous phase in 10ml deionized waters.In the stirring 10~20 minutes of high speed magneton (1000rpm), oil phase is dispersed to company
Emulsion is formed in continuous aqueous phase.By adjusting the time of PVA content, the ratio of oil phase/water phase, and magneton stirring, control
1.0~1.5 μm of emulsion diameter.
Step 3, synthesis PEG-co-AA microgels core (2).The emulsion being made is placed in 100w lower 20 minutes of uviol lamp.
Ultraviolet excitation light trigger produces free radical, under the principle of radical polymerization, the double bond quilt of PEGDA and AA in oil droplet
Open, cause chain to increase the cross-linking reaction between chain.Therefore, the PEGDA and AA in oil droplet form microgel core, with 70% second
The method of alcohol suspension-centrifugation is purified repeatedly for three times, and the method suspend-centrifuged with deionized water removes alcohol.Pass through control
PEGDA and AA processed ratio 10:0.1~1, the charge density of microgel core is controlled, the minus 6~40mv of potential range is formed.It is made
Protomere core collected by 16000~50000r/min high speed centrifugations, with the filter membrane of different pore size can selectivity obtain
Required 1~1000nm of particle size, the protomere core being finally collected into is dispersed in 10mlPBS phosphate buffers.
Step 4, PEG-co-AA microgels core surface modification PMAA high charge densities shell (2).Microgel core after purification
(0.2mg/ in polyvinylpyrrolidone (poly (N-vinylpyrrolidone), PVPON) PBS solution is dispersed to first
ML, pH 2,0.01M PBS), after 15 minutes, by microgel grain by filtering, the mode of centrifugation is cleaned.Then, by PVPON
The micelle managed is dispersed in polymethylacrylic acid PMAA PBS solution (0.2mg/mL, pH 2,0.01M PBS), 15 minutes
Afterwards, clean in aforementioned manners.Repeat above method 5~30 times, be formed with 5~30 layers of PVPON/PMAA gels of hydrogen bond crosslinks
Layer.
Step 5, cross-linked stable PMAA gels shell (2).Micro-gel particles are dispersed in 1- ethyls-(3- dimethylaminos
Propyl group) phosphinylidyne diimmonium salt hydrochlorate 5mg/mL and hydroxy thiosuccinimide 5mg/mL 0.01M PBS solutions (PH5) in
1.5 hour；Afterwards, micelle is separated from solution, and be dipped into 10mg/mL ethylenediamine (ethylenediamine,
EDA) the covalent cross-linking that 14 hours are carried out between PMAA layers in solution (pH5.8)；Finally, in order to remove PVPON and unreacted
Other reagents, PEG-co-AA/PMAA composite microgels grain is soaked in pH7.5 0.01M PBS 2 hours, centrifugation-filtering
Composite microgel grain is suspended in PBS (pH7.4,0.1M) afterwards.Exist using dynamic light scattering and Zeta electric potential assay method
The size of composite microgel and powered situation in the case of different pH4~9 and ionic strength.Compound protomere (2) diameter 100~
400 μm, 0.6~8 μm of altitude range.The PMAA microgels layer minus 6~0mv of potential range, 5~30 layers of the number of plies.The compound protomere
Possesses the characteristic that electric charge is shifted automatically from high potential to low potential.
Step 6, the structure (3) on the topological antibacterial structure surface of titanium.Deposition of the microgel on titanium surface:Compound protomere is hanged
In the PBS for floating over 0.01M, pH7.5, regulation micelle concentration is 1.0~2.0 × 1010To 1011Individual/ml.By many of positively charged
PLL positively charged macromolecule layer titanium sheet is dipped into above-mentioned microgel suspension, and due to electrostatic adsorption, microgel will
Automatic absorbing is to titanium plate surface.After 0.5~10 hour, titanium sheet is removed, and is alternately rinsed with 0.01M PBS and deionized water,
Dry.The titanium sheet prepared is observed under ESEM, confirms the absorption of microgel.Build and screen gel diameter range
100~400 μm, the antibacterial topological structure surface titanium plate of 0.5~4 μm of 0.6~8 μm of altitude range and gel interstice coverage.
Step 7, beta-alexin -1~3, lactoferrin 1-11, LL-37, the similar body (histatin of histatins
Analogue DHVAR-5), protegrins -1 (Protegrins-1) and artificial synthesized cationic antibacterial peptide
It is loaded into the titanium surface (4) of microgel modification.Under the conditions of 36~37 DEG C, the titanium surface of dry composite microgel modification is soaked
It is during 10~20mL, concentration are the hBD-3 or lactoferrin 1-11PBS solution that 1mg/mL, pH value are 7.4, due to band in concentration
Electrical property is on the contrary, hBD-3 is loaded into composite microgel.Form the load beta-alexin -1~3 with electrical potential difference, newborn iron egg
White 1-11, LL-37, the similar body of histatins (histatin analogue DHVAR-5), protegrins -1
(Protegrins-1) and the topological antibacterial structure surface recombination protomere layer (4) of artificial synthesized cationic antibacterial peptide, at room temperature
Dry, obtain intelligent nano-hydrogel topological structure antibacterial bone plate.
Intellectuality nano-hydrogel topological structure antibacterial bone plate of the invention is through internal (NZw) and external medicine
Kinetic measurement is confirmed, is constructed a diameter of 200~300nm PEG-co-AA protomeres with loading cation antibacterial peptide, is allowed to
Play in the case that bacterium is present in implants surface, it is used as stimulus " contact sterilization " (contact-
Killing intelligent stimuli responsive mechanism), is formed with fixed fracture site and strongly anti-infective and suppression bacterial biof iotalm
Effect, can locally maintain effective antimicrobial concentration of 10 days.
Beneficial effect：Emulsion polymerization technology, multilayer polymeric technology in Applied Surface Chemistry technology of the present invention, electrostatic precipitation
Technology prepares intelligent nano-hydrogel topological structure antibacterial bone plate, not only breaches half-life short in the antibacterial peptide aqueous solution
" bottleneck ", it is important to be allowed to play in the case that bacterium is present in implants surface, as stimulus, " contact is killed for it
The intelligent stimuli responsive mechanism of bacterium " (contact-killing).Conversely, as bacterium be not present when, then retain antibacterial peptide in connecing
Plate surface.And changing the surface topography and chemical constitution of biomaterial simultaneously, its three-dimensional topology table face is highly beneficial
In the tactophily and propagation of Gegenbaur's cell.So as to realize that Gegenbaur's cell etc. is sticked in interior plant surface fast-growth and reduction bacterium
Attached and utilization antibacterial peptide killing bacterium, causes " target spot " control of formation really suppression bacterial biof iotalm to originate rank at it
Section, finally to reduce the incidence of " implants infections relating ".Compared with other slow release methods, the technology is led to (1) is easy to
The electrostatic adsorption crossed between microgel and matrix quickly and easily builds surface topology；(2) keep anti-by ionic bond
The structure and function-stable of the small molecule peptide or protein such as bacterium peptide；(3) by controlling between metal material surface roughness and protomere
Away from, in favor of bone or fibroblast growth propagation and " intellectuality " advantage for suppressing bacterial adhesion growth and biofilm formation,
Meet " the third strategy " of preventing and treating " fixture infections relating " at present and the development trend of " smart material ".Directly suppress life
The formation and development of thing film, obtain preventing and treating ODRI curative effect.Traditionally taboo is broken through, to bone caused by ODRI and open or firearm injury
Folding into the row I phases repairs and prevents and treats the generation of Traumatic osteomyelitis, development, shortens the course for the treatment of so as to reach, significantly improves the mesh of curative effect
's.The present invention not only has fixed fracture site and feature that is strongly anti-infective and suppressing bacterial biof iotalm formation, and possesses
" intellectuality " is distributed antibacterial peptide and bacterium as stimulus to low potential region from high potential region and just played " contact automatically
Property sterilization " intelligent stimuli responsive mechanism.
Brief description of the drawings
Fig. 1 is the preparation flow and effect schematic diagram of bone plate of the present invention；
Embodiment
Embodiment 1
The manufacture method of intelligent nano-hydrogel topological structure antibacterial bone plate, is carried out as follows：
Step 1, titanium or (1) surface preparation of titanium alloy bone plate and titanium surface on-line treatment.Titanium or titanium are closed
Golden bone plate (1) is cleaned by ultrasonic with distilled water, acetone, 75% ethanol, distilled water successively, panel carries out hydrophilic treated
Each 10~20 minutes, 0.2mg/mL is inserted, mean molecule quantity is 30~50k, pH 9 many PLL (poly-l-
Lysine, PLL) 2~3 hours, in titanium alloy substrate formation positively charged macromolecule layer, initial layer deposition is carried out, is used after taking-up
Deionized water is cleaned, and is dried at room temperature for stand-by.
Step 2, PEGDA/AA emulsions are prepared.Make even respectively the polyethylene glycol acrylate of average molecular weight 500~600
(Polyethylene Glycol Diacrylate, PEGDA) 200 μ l, acrylic monomers (acrylic acid, AA) is at 50 DEG C
0~20 μ l, and the μ l of light trigger (Darocur 1173, Ciba) 10 are left and taken after distillation, co-dissolve is in dichloromethane
Oil phase is formed in (dichloromethane, DCM) 1ml.1~10% polyvinyl alcohol (PVA) 1~3ml is molten as emulsifying agent
Solution forms aqueous phase in 10ml deionized waters.In the stirring 10~20 minutes of high speed magneton (1000rpm), oil phase is dispersed to company
Emulsion is formed in continuous aqueous phase.By adjusting the time of PVA content, the ratio of oil phase/water phase, and magneton stirring, control
1.0~1.5 μm of emulsion diameter.
Step 3, PEG-co-AA microgel cores are synthesized.The emulsion being made is placed in 100w lower 20 minutes of uviol lamp.It is ultraviolet
Phot-luminescence initiator produces free radical, and under the principle of radical polymerization, the double bond of PEGDA and AA in oil droplet are beaten
Open, cause chain to increase the cross-linking reaction between chain.Therefore, the PEGDA and AA in oil droplet form microgel core, with 70% ethanol
The method of suspension-centrifugation is purified repeatedly for three times, and the method suspend-centrifuged with deionized water removes alcohol.Pass through control
PEGDA and AA ratio 10:0.1~1, the charge density of microgel core is controlled, the minus 6~40mv of potential range is formed.It is made
Protomere core is collected by 16000~50000r/min high speed centrifugations, with the filter membrane of different pore size can selectivity obtain institute
1~the 1000nm of particle size needed, the protomere core being finally collected into is dispersed in 10mlPBS phosphate buffers.
Step 4, PEG-co-AA microgels core surface modification PMAA high charge density shells.Microgel core after purification is first
First be dispersed in polyvinylpyrrolidone (poly (N-vinylpyrrolidone), PVPON) PBS solution (0.2mg/mL,
PH 2,0.01M PBS), after 15 minutes, by microgel grain by filtering, the mode of centrifugation is cleaned.Then, PVPON is treated
Micelle be dispersed in polymethylacrylic acid PMAA PBS solution (0.2mg/mL, pH 2,0.01M PBS), after 15 minutes, use
The above method is cleaned.Repeat above method 5~30 times, be formed with 5~30 layers of PVPON/PMAA gel layers of hydrogen bond crosslinks.
Step 5, cross-linked stable PMAA gels shell.Micro-gel particles are dispersed in 1- ethyls-(3- dimethylaminos third
Base) phosphinylidyne diimmonium salt hydrochlorate 5mg/mL and hydroxy thiosuccinimide 5mg/mL 0.01M PBS solutions (PH5) in 1.5
Hour；Afterwards, micelle is separated from solution, and it is molten to be dipped into 10mg/mL ethylenediamine (ethylenediamine, EDA)
The covalent cross-linking that 14 hours are carried out between PMAA layers in liquid (pH5.8)；Finally, tried to remove PVPON and unreacted other
Agent, PEG-co-AA/PMAA composite microgels grain is soaked in pH7.5 0.01M PBS 2 hours, multiple after centrifugation-filtering
Microgel grain is closed to be suspended in PBS (pH7.4,0.1M).Using dynamic light scattering and Zeta electric potential assay method in different pH4
~9 and ionic strength in the case of composite microgel size and powered situation.100~400 μm of 2 diameter of compound protomere, height
Spend 0.6~8 μm of scope.The PMAA microgels layer minus 6~0mv of potential range, 5~30 layers of the number of plies.The compound protomere 2 possesses electric charge
The characteristic shifted automatically from high potential to low potential.
Step 6, the structure on the topological antibacterial structure surface of titanium.Deposition of the microgel on titanium surface:Compound protomere 2 is suspended
In 0.01M, pH7.5 PBS, regulation micelle concentration is 1.0~2.0 × 1010To 1011Individual/ml.By the poly L of positively charged
Lysine positively charged macromolecule layer titanium sheet is dipped into above-mentioned microgel suspension, and due to electrostatic adsorption, microgel will be certainly
It is dynamic to be adsorbed onto titanium plate surface.After 0.5~10 hour, titanium sheet is removed, and is alternately rinsed, done with 0.01M PBS and deionized water
It is dry.The titanium sheet prepared is observed under ESEM, confirms the absorption of microgel.Build and screen gel diameter range
100~400 μm, the antibacterial topological structure surface titanium plate of 0.5~4 μm of 0.6~8 μm of altitude range and gel interstice coverage.
Step 7, beta-alexin -1~3 are loaded into the titanium surface of microgel modification.Under the conditions of 36~37 DEG C, that dries answers
Close microgel modification titanium surface be soaked in concentration be 10~20mL, concentration be 1mg/mL, pH value be 7.4 hBD-1~
In 3PBS solution, due to powered property on the contrary, hBD-1~3 are loaded into composite microgel.Form the load with electrical potential difference
The topological antibacterial structure surface recombination protomere layer in beta-alexin -1~3, dries, obtains intelligent nano-hydrogel and open up at room temperature
Flutter structure antibacterial bone plate.
Step 8, antibacterial peptide sustained release steel plate step 7 obtained is placed in 40 ° of incubators, after solidifying 10 minutes, encapsulation, epoxy
Ethane is sterilized.
Finally obtain as shown in figure 1, intelligent nano-hydrogel topological structure antibacterial bone plate, the titanium bone plate
The topological antibacterial structure surface recombination protomere 2 of titanium or the titanium alloy bone plate loads the unit plane of beta-alexin -1~3
0.025~1mg/mL of product concentration.Titanium or titanium alloy synthetism the plate surface covering is interior by PEG-co-AA microgel cores
Core, outer layer controlled release shell is combined micro- for the intelligent charge gradient radial distribution of pH responsive type polymethylacrylic acid PMAA microgels layer
Micelle 2,100~400 μm of 2 coating diameter of compound protomere, 0.6~8 μm of the altitude range.PEG-co-AA microgel nuclear power gesture
Minus 6~the 40mv of scope, 1~1000nm of core internal diameter；The PMAA microgels layer minus 6~0mv of potential range, 5~30 layers of the number of plies.This is combined
Protomere 2 possesses the characteristic that electric charge is shifted automatically from high potential to low potential.Titanium or titanium alloy the bone plate topology is anti-
Bacterium body structure surface is combined 0.5~4 μm of 2 spacing range of protomere.
Embodiment 2
The manufacture method of intelligent nano-hydrogel topological structure antibacterial bone plate, is carried out as follows：
Step 1, titanium or (1) surface preparation of titanium alloy bone plate and titanium surface on-line treatment.Titanium or titanium are closed
Golden bone plate (1) is cleaned by ultrasonic with distilled water, acetone, 75% ethanol, distilled water successively, panel carries out hydrophilic treated
Each 10~20 minutes, 0.2mg/mL is inserted, mean molecule quantity is 30~50k, pH 9 many PLL (poly-l-
Lysine, PLL) 2~3 hours, in titanium alloy substrate formation positively charged macromolecule layer, initial layer deposition is carried out, is used after taking-up
Deionized water is cleaned, and is dried at room temperature for stand-by.
Step 2, PEGDA/AA emulsions are prepared.Make even respectively the polyethylene glycol acrylate of average molecular weight 500~600
(Polyethylene Glycol Diacrylate, PEGDA) 200 μ l, acrylic monomers (acrylic acid, AA) is at 50 DEG C
0~20 μ l, and the μ l of light trigger (Darocur 1173, Ciba) 10 are left and taken after distillation, co-dissolve is in dichloromethane
Oil phase is formed in (dichloromethane, DCM) 1ml.1~10% polyvinyl alcohol (PVA) 1~3ml is molten as emulsifying agent
Solution forms aqueous phase in 10ml deionized waters.In the stirring 10~20 minutes of high speed magneton (1000rpm), oil phase is dispersed to company
Emulsion is formed in continuous aqueous phase.By adjusting the time of PVA content, the ratio of oil phase/water phase, and magneton stirring, control
1.0~1.5 μm of emulsion diameter.
Step 3, PEG-co-AA microgel cores are synthesized.The emulsion being made is placed in 100w lower 20 minutes of uviol lamp.It is ultraviolet
Phot-luminescence initiator produces free radical, and under the principle of radical polymerization, the double bond of PEGDA and AA in oil droplet are beaten
Open, cause chain to increase the cross-linking reaction between chain.Therefore, the PEGDA and AA in oil droplet form microgel core, with 70% ethanol
The method of suspension-centrifugation is purified repeatedly for three times, and the method suspend-centrifuged with deionized water removes alcohol.Pass through control
PEGDA and AA ratio 10:0.1~1, the charge density of microgel core is controlled, the minus 6~40mv of potential range is formed.It is made
Protomere core is collected by 16000~50000r/min high speed centrifugations, with the filter membrane of different pore size can selectivity obtain institute
1~the 1000nm of particle size needed, the protomere core being finally collected into is dispersed in 10mlPBS phosphate buffers.
Step 4, PEG-co-AA microgels core surface modification PMAA high charge density shells.Microgel core after purification is first
First be dispersed in polyvinylpyrrolidone (poly (N-vinylpyrrolidone), PVPON) PBS solution (0.2mg/mL,
PH 2,0.01M PBS), after 15 minutes, by microgel grain by filtering, the mode of centrifugation is cleaned.Then, PVPON is treated
Micelle be dispersed in polymethylacrylic acid PMAA PBS solution (0.2mg/mL, pH 2,0.01M PBS), after 15 minutes, use
The above method is cleaned.Repeat above method 5~30 times, be formed with 5~30 layers of PVPON/PMAA gel layers of hydrogen bond crosslinks.
Step 5, cross-linked stable PMAA gels shell.Micro-gel particles are dispersed in 1- ethyls-(3- dimethylaminos third
Base) phosphinylidyne diimmonium salt hydrochlorate 5mg/mL and hydroxy thiosuccinimide 5mg/mL 0.01M PBS solutions (PH5) in 1.5
Hour；Afterwards, micelle is separated from solution, and it is molten to be dipped into 10mg/mL ethylenediamine (ethylenediamine, EDA)
The covalent cross-linking that 14 hours are carried out between PMAA layers in liquid (pH5.8)；Finally, tried to remove PVPON and unreacted other
Agent, PEG-co-AA/PMAA composite microgels grain is soaked in pH7.5 0.01M PBS 2 hours, multiple after centrifugation-filtering
Microgel grain is closed to be suspended in PBS (pH7.4,0.1M).Using dynamic light scattering and Zeta electric potential assay method in different pH4
~9 and ionic strength in the case of composite microgel size and powered situation.100~400 μm of 2 diameter of compound protomere, height
Spend 0.6~8 μm of scope.The PMAA microgels layer minus 6~0mv of potential range, 5~30 layers of the number of plies.The compound protomere 2 possesses electric charge
The characteristic shifted automatically from high potential to low potential.
Step 6, the structure on the topological antibacterial structure surface of titanium.Deposition of the microgel on titanium surface:Compound protomere 2 is suspended
In 0.01M, pH7.5 PBS, regulation micelle concentration is 1.0~2.0 × 1010To 1011Individual/ml.By the poly L of positively charged
Lysine positively charged macromolecule layer titanium sheet is dipped into above-mentioned microgel suspension, and due to electrostatic adsorption, microgel will be certainly
It is dynamic to be adsorbed onto titanium plate surface.After 0.5~10 hour, titanium sheet is removed, and is alternately rinsed, done with 0.01M PBS and deionized water
It is dry.The titanium sheet prepared is observed under ESEM, confirms the absorption of microgel.Build and screen gel diameter range
100~400 μm, the antibacterial topological structure surface titanium plate of 0.5~4 μm of 0.6~8 μm of altitude range and gel interstice coverage.
Step 7, LL-37 is loaded into the titanium surface of microgel modification.Under the conditions of 36~37 DEG C, dry composite microgel is repaiied
The titanium surface of decorations is soaked in concentration, due to
Powered property is on the contrary, LL-37 is loaded into composite microgel.Form the topological antibacterial structure surfaces of the LL-37 with electrical potential difference
Compound protomere layer, dries, obtains intelligent nano-hydrogel topological structure antibacterial bone plate at room temperature.
Step 8, antibacterial peptide sustained release steel plate step 7 obtained is placed in 40 ° of incubators, after solidifying 10 minutes, encapsulation, epoxy
Ethane is sterilized.
Finally obtain as shown in figure 1, intelligent nano-hydrogel topological structure antibacterial bone plate, the titanium bone plate
The topological antibacterial structure surface recombination protomere 2 of titanium or the titanium alloy bone plate loads LL-37 per unit area concentrations 0.025
~1mg/mL.Titanium or titanium alloy synthetism the plate surface covering is kernel, outer layer controlled release shell by PEG-co-AA microgels core
Intelligent charge gradient radial distribution for pH responsive type polymethylacrylic acid PMAA microgels layer is combined protomere 2, and this is combined
100~400 μm of 2 coating diameter of protomere, 0.6~8 μm of altitude range.PEG-co-AA microgels core potential range minus 6~
40mv, 1~1000nm of core internal diameter；The PMAA microgels layer minus 6~0mv of potential range, 5~30 layers of the number of plies.The compound protomere 2 has
The characteristic that standby electric charge is shifted automatically from high potential to low potential.The topological antibacterial structure surface of titanium or the titanium alloy bone plate
0.5~4 μm of 2 spacing range of compound protomere.
Embodiment 3
The manufacture method of intelligent nano-hydrogel topological structure antibacterial bone plate, is carried out as follows：
Step 1, titanium or (1) surface preparation of titanium alloy bone plate and titanium surface on-line treatment.Titanium or titanium are closed
Golden bone plate (1) is cleaned by ultrasonic with distilled water, acetone, 75% ethanol, distilled water successively, panel carries out hydrophilic treated
Each 10~20 minutes, 0.2mg/mL is inserted, mean molecule quantity is 30~50k, pH 9 many PLL (poly-l-
Lysine, PLL) 2~3 hours, in titanium alloy substrate formation positively charged macromolecule layer, initial layer deposition is carried out, is used after taking-up
Deionized water is cleaned, and is dried at room temperature for stand-by.
Step 2, PEGDA/AA emulsions are prepared.Make even respectively the polyethylene glycol acrylate of average molecular weight 500~600
(Polyethylene Glycol Diacrylate, PEGDA) 200 μ l, acrylic monomers (acrylic acid, AA) is at 50 DEG C
0~20 μ l, and the μ l of light trigger (Darocur 1173, Ciba) 10 are left and taken after distillation, co-dissolve is in dichloromethane
Oil phase is formed in (dichloromethane, DCM) 1ml.1~10% polyvinyl alcohol (PVA) 1~3ml is molten as emulsifying agent
Solution forms aqueous phase in 10ml deionized waters.In the stirring 10~20 minutes of high speed magneton (1000rpm), oil phase is dispersed to company
Emulsion is formed in continuous aqueous phase.By adjusting the time of PVA content, the ratio of oil phase/water phase, and magneton stirring, control
1.0~1.5 μm of emulsion diameter.
Step 3, PEG-co-AA microgel cores are synthesized.The emulsion being made is placed in 100w lower 20 minutes of uviol lamp.It is ultraviolet
Phot-luminescence initiator produces free radical, and under the principle of radical polymerization, the double bond of PEGDA and AA in oil droplet are beaten
Open, cause chain to increase the cross-linking reaction between chain.Therefore, the PEGDA and AA in oil droplet form microgel core, with 70% ethanol
The method of suspension-centrifugation is purified repeatedly for three times, and the method suspend-centrifuged with deionized water removes alcohol.Pass through control
PEGDA and AA ratio 10:0.1~1, the charge density of microgel core is controlled, the minus 6~40mv of potential range is formed.It is made
Protomere core is collected by 16000~50000r/min high speed centrifugations, with the filter membrane of different pore size can selectivity obtain institute
1~the 1000nm of particle size needed, the protomere core being finally collected into is dispersed in 10mlPBS phosphate buffers.
Step 4, PEG-co-AA microgels core surface modification PMAA high charge density shells.Microgel core after purification is first
First be dispersed in polyvinylpyrrolidone (poly (N-vinylpyrrolidone), PVPON) PBS solution (0.2mg/mL,
PH 2,0.01M PBS), after 15 minutes, by microgel grain by filtering, the mode of centrifugation is cleaned.Then, PVPON is treated
Micelle be dispersed in polymethylacrylic acid PMAA PBS solution (0.2mg/mL, pH 2,0.01M PBS), after 15 minutes, use
The above method is cleaned.Repeat above method 5~30 times, be formed with 5~30 layers of PVPON/PMAA gel layers of hydrogen bond crosslinks.
Step 5, cross-linked stable PMAA gels shell.Micro-gel particles are dispersed in 1- ethyls-(3- dimethylaminos third
Base) phosphinylidyne diimmonium salt hydrochlorate 5mg/mL and hydroxy thiosuccinimide 5mg/mL 0.01M PBS solutions (PH5) in 1.5
Hour；Afterwards, micelle is separated from solution, and it is molten to be dipped into 10mg/mL ethylenediamine (ethylenediamine, EDA)
The covalent cross-linking that 14 hours are carried out between PMAA layers in liquid (pH5.8)；Finally, tried to remove PVPON and unreacted other
Agent, PEG-co-AA/PMAA composite microgels grain is soaked in pH7.5 0.01M PBS 2 hours, multiple after centrifugation-filtering
Microgel grain is closed to be suspended in PBS (pH7.4,0.1M).Using dynamic light scattering and Zeta electric potential assay method in different pH4
~9 and ionic strength in the case of composite microgel size and powered situation.100~400 μm of 2 diameter of compound protomere, height
Spend 0.6~8 μm of scope.The PMAA microgels layer minus 6~0mv of potential range, 5~30 layers of the number of plies.The compound protomere 2 possesses electric charge
The characteristic shifted automatically from high potential to low potential.
Step 6, the structure on the topological antibacterial structure surface of titanium.Deposition of the microgel on titanium surface:Compound protomere 2 is suspended
In 0.01M, pH7.5 PBS, regulation micelle concentration is 1.0~2.0 × 1010To 1011Individual/ml.By the poly L of positively charged
Lysine positively charged macromolecule layer titanium sheet is dipped into above-mentioned microgel suspension, and due to electrostatic adsorption, microgel will be certainly
It is dynamic to be adsorbed onto titanium plate surface.After 0.5~10 hour, titanium sheet is removed, and is alternately rinsed, done with 0.01M PBS and deionized water
It is dry.The titanium sheet prepared is observed under ESEM, confirms the absorption of microgel.Build and screen gel diameter range
100~400 μm, the antibacterial topological structure surface titanium plate of 0.5~4 μm of 0.6~8 μm of altitude range and gel interstice coverage.
Step 7, the similar body of histatins (histatin analogue DHVAR-5) is loaded into the titanium table of microgel modification
Face.Under the conditions of 36~37 DEG C, it is that 10~20mL, concentration are that the titanium surface of dry composite microgel modification, which is soaked in concentration,
1mg/mL, pH value for 7.4 hBD-3PBS solution in, due to powered property on the contrary, the similar body (histatin of histatins
Analogue DHVAR-5) it is loaded into composite microgel.Form the similar body (histatin of histatins with electrical potential difference
Analogue DHVAR-5) topological antibacterial structure surface recombination protomere layer, dry at room temperature, obtain intelligent nano-hydrogel
Topological structure antibacterial bone plate.
Step 8, antibacterial peptide sustained release steel plate step 7 obtained is placed in 40 ° of incubators, after solidifying 10 minutes, encapsulation, epoxy
Ethane is sterilized.
Finally obtain as shown in figure 1, intelligent nano-hydrogel topological structure antibacterial bone plate, the titanium bone plate
It is dense that the topological antibacterial structure surface recombination protomere 2 of titanium or the titanium alloy bone plate loads the unit area of beta-alexin -3
Spend 0.025~1mg/mL.Titanium or titanium alloy synthetism the plate surface covering is kernel by PEG-co-AA microgels core, outside
Layer controlled release shell is that the intelligent charge gradient radial distribution of pH responsive type polymethylacrylic acid PMAA microgels layer is combined protomere
2,100~400 μm of 2 coating diameter of compound protomere, 0.6~8 μm of the altitude range.PEG-co-AA microgel core potential ranges
Minus 6~40mv, 1~1000nm of core internal diameter；The PMAA microgels layer minus 6~0mv of potential range, 5~30 layers of the number of plies.This is combined micro- glue
Grain 2 possesses the characteristic that electric charge is shifted automatically from high potential to low potential.The topological antibacterial knot of titanium or the titanium alloy bone plate
0.5~4 μm of 2 spacing range of structure surface recombination protomere.
Embodiment 4
The manufacture method of intelligent nano-hydrogel topological structure antibacterial bone plate, is carried out as follows：
Step 1, titanium or (1) surface preparation of titanium alloy bone plate and titanium surface on-line treatment.Titanium or titanium are closed
Golden bone plate (1) is cleaned by ultrasonic with distilled water, acetone, 75% ethanol, distilled water successively, panel carries out hydrophilic treated
Each 10~20 minutes, 0.2mg/mL is inserted, mean molecule quantity is 30~50k, pH 9 many PLL (poly-l-
Lysine, PLL) 2~3 hours, in titanium alloy substrate formation positively charged macromolecule layer, initial layer deposition is carried out, is used after taking-up
Deionized water is cleaned, and is dried at room temperature for stand-by.
Step 2, PEGDA/AA emulsions are prepared.Make even respectively the polyethylene glycol acrylate of average molecular weight 500~600
(Polyethylene Glycol Diacrylate, PEGDA) 200 μ l, acrylic monomers (acrylic acid, AA) is at 50 DEG C
0~20 μ l, and the μ l of light trigger (Darocur 1173, Ciba) 10 are left and taken after distillation, co-dissolve is in dichloromethane
Oil phase is formed in (dichloromethane, DCM) 1ml.1~10% polyvinyl alcohol (PVA) 1~3ml is molten as emulsifying agent
Solution forms aqueous phase in 10ml deionized waters.In the stirring 10~20 minutes of high speed magneton (1000rpm), oil phase is dispersed to company
Emulsion is formed in continuous aqueous phase.By adjusting the time of PVA content, the ratio of oil phase/water phase, and magneton stirring, control
1.0~1.5 μm of emulsion diameter.
Step 3, PEG-co-AA microgel cores are synthesized.The emulsion being made is placed in 100w lower 20 minutes of uviol lamp.It is ultraviolet
Phot-luminescence initiator produces free radical, and under the principle of radical polymerization, the double bond of PEGDA and AA in oil droplet are beaten
Open, cause chain to increase the cross-linking reaction between chain.Therefore, the PEGDA and AA in oil droplet form microgel core, with 70% ethanol
The method of suspension-centrifugation is purified repeatedly for three times, and the method suspend-centrifuged with deionized water removes alcohol.Pass through control
PEGDA and AA ratio 10:0.1~1, the charge density of microgel core is controlled, the minus 6~40mv of potential range is formed.It is made
Protomere core is collected by 16000~50000r/min high speed centrifugations, with the filter membrane of different pore size can selectivity obtain institute
1~the 1000nm of particle size needed, the protomere core being finally collected into is dispersed in 10mlPBS phosphate buffers.
Step 4, PEG-co-AA microgels core surface modification PMAA high charge density shells.Microgel core after purification is first
First be dispersed in polyvinylpyrrolidone (poly (N-vinylpyrrolidone), PVPON) PBS solution (0.2mg/mL,
PH 2,0.01M PBS), after 15 minutes, by microgel grain by filtering, the mode of centrifugation is cleaned.Then, PVPON is treated
Micelle be dispersed in polymethylacrylic acid PMAA PBS solution (0.2mg/mL, pH 2,0.01M PBS), after 15 minutes, use
The above method is cleaned.Repeat above method 5~30 times, be formed with 5~30 layers of PVPON/PMAA gel layers of hydrogen bond crosslinks.
Step 5, cross-linked stable PMAA gels shell.Micro-gel particles are dispersed in 1- ethyls-(3- dimethylaminos third
Base) phosphinylidyne diimmonium salt hydrochlorate 5mg/mL and hydroxy thiosuccinimide 5mg/mL 0.01M PBS solutions (PH5) in 1.5
Hour；Afterwards, micelle is separated from solution, and it is molten to be dipped into 10mg/mL ethylenediamine (ethylenediamine, EDA)
The covalent cross-linking that 14 hours are carried out between PMAA layers in liquid (pH5.8)；Finally, tried to remove PVPON and unreacted other
Agent, PEG-co-AA/PMAA composite microgels grain is soaked in pH7.5 0.01M PBS 2 hours, multiple after centrifugation-filtering
Microgel grain is closed to be suspended in PBS (pH7.4,0.1M).Using dynamic light scattering and Zeta electric potential assay method in different pH4
~9 and ionic strength in the case of composite microgel size and powered situation.100~400 μm of 2 diameter of compound protomere, height
Spend 0.6~8 μm of scope.The PMAA microgels layer minus 6~0mv of potential range, 5~30 layers of the number of plies.The compound protomere 2 possesses electric charge
The characteristic shifted automatically from high potential to low potential.
Step 6, the structure on the topological antibacterial structure surface of titanium.Deposition of the microgel on titanium surface:Compound protomere 2 is suspended
In 0.01M, pH7.5 PBS, regulation micelle concentration is 1.0~2.0 × 1010To 1011Individual/ml.By the poly L of positively charged
Lysine positively charged macromolecule layer titanium sheet is dipped into above-mentioned microgel suspension, and due to electrostatic adsorption, microgel will be certainly
It is dynamic to be adsorbed onto titanium plate surface.After 0.5~10 hour, titanium sheet is removed, and is alternately rinsed, done with 0.01M PBS and deionized water
It is dry.The titanium sheet prepared is observed under ESEM, confirms the absorption of microgel.Build and screen gel diameter range
100~400 μm, the antibacterial topological structure surface titanium plate of 0.5~4 μm of 0.6~8 μm of altitude range and gel interstice coverage.
Step 7, beta-alexin -1 is loaded into the titanium surface of microgel modification.Under the conditions of 36~37 DEG C, that dries is compound
It is that 10~20mL, concentration are the lactoferrin 1- that 1mg/mL, pH value are 7.4 that the titanium surface of microgel modification, which is soaked in concentration,
In 11PBS solution, due to powered property on the contrary, lactoferrin 1-11 is loaded into composite microgel.Being formed has electrical potential difference
The topological antibacterial structure surface recombination protomere layers of lactoferrin 1-11, dry at room temperature, obtain intelligent nano-hydrogel and open up
Flutter structure antibacterial bone plate.
Step 8, antibacterial peptide sustained release steel plate step 7 obtained is placed in 40 ° of incubators, after solidifying 10 minutes, encapsulation, epoxy
Ethane is sterilized.
Finally obtain as shown in figure 1, intelligent nano-hydrogel topological structure antibacterial bone plate, the titanium bone plate
It is dense that the topological antibacterial structure surface recombination protomere 2 of titanium or the titanium alloy bone plate loads lactoferrin 1-11 unit areas
Spend 0.025~1mg/mL.Titanium or titanium alloy synthetism the plate surface covering is kernel by PEG-co-AA microgels core, outside
Layer controlled release shell is that the intelligent charge gradient radial distribution of pH responsive type polymethylacrylic acid PMAA microgels layer is combined protomere
2,100~400 μm of 2 coating diameter of compound protomere, 0.6~8 μm of the altitude range.PEG-co-AA microgel core potential ranges
Minus 6~40mv, 1~1000nm of core internal diameter；The PMAA microgels layer minus 6~0mv of potential range, 5~30 layers of the number of plies.This is combined micro- glue
Grain 2 possesses the characteristic that electric charge is shifted automatically from high potential to low potential.The topological antibacterial knot of titanium or the titanium alloy bone plate
0.5~4 μm of structure surface recombination protomere spacing range.
Claims (5)
1. a kind of intelligent nano-hydrogel topological structure antibacterial bone plate, including bone plate (1), it is characterised in that：It is described to connect
The covering of hone lamella (1) surface is kernel by polyethylene glycol-co- acroleic acid microgels core, and outer layer controlled release shell is the poly- methyl of pH responsive types
The intelligent charge gradient radial distribution of acroleic acid microgel layer is combined protomere (2), the compound protomere coating stainless steel,
Titanium or the compound protomere (2) on the topological antibacterial structure surface of titanium alloy bone plate (1) formation, the compound protomere (2)
100~400 μm of diameter, 0.6~8 μm of altitude range；Polyethylene glycol-co- acroleic acid microgel minus 6~the 40mv of core potential range,
1~1000nm of core internal diameter；The pH responsive type polymethylacrylic acid microgel layer minus 6~0mv of potential range, 5~30 layers of the number of plies.
2. intelligent nano-hydrogel topological structure antibacterial bone plate according to claim 1, it is characterised in that：The synthetism
Compound protomere (2) spacing range on plate (1) upper topology antibacterial structure surface is 0.5~4 μm.
3. intelligent nano-hydrogel topological structure antibacterial bone plate according to claim 1, it is characterised in that：The synthetism
Plate (1) upper topology antibacterial structure surface compound protomere (2) load beta-alexin -1~3, lactoferrin 1-11, LL-37,
The similar body of histatins, protegrins -1 and artificial synthesized cationic antibacterial peptide.
4. intelligent nano-hydrogel topological structure antibacterial bone plate according to claim 3, it is characterised in that：The synthetism
Plate (1) upper topology antibacterial structure surface compound protomere (2) load beta-alexin -1~3, lactoferrin 1-11, LL-37,
The similar body of histatins, protegrins -1 and artificial synthesized cationic antibacterial peptide per unit area concentration are
0.025~1mg/mL.
5. a kind of manufacture method of intelligent nano-hydrogel topological structure antibacterial bone plate as claimed in claim 1, its feature
It is：Carry out as follows：
Step 1, titanium or (1) surface preparation of titanium alloy bone plate and titanium surface on-line treatment, titanium or titanium alloy connect
Hone lamella (1) is cleaned by ultrasonic with distilled water, acetone, 75% ethanol, distilled water successively, panel carries out hydrophilic treated each 10
~20 minutes, 0.2mg/mL is inserted, mean molecule quantity is 30~50k, pH9 many PLLs 2~3 hours, titanium alloy-based
Body formation positively charged macromolecule layer, carries out initial layer deposition, is washed with deionized water only, is dried at room temperature for stand-by after taking-up；
Step 2, PEGDA/AA emulsions are prepared, the polyethylene glycol acrylate 200 μ l for average molecular weight 500~600 of making even respectively, third
Acrylic monomer leaves and takes 0~20 μ l, and the μ l of light trigger 10 after being distilled at 50 DEG C, and co-dissolve is formed in dichloromethane 1ml
Oil phase；1~10% 1~3ml of polyvinyl alcohol is dissolved in 10ml deionized waters as emulsifying agent and forms aqueous phase；In high speed magnetic
Son is with 1000rpm stirring 10~20 minutes, and oil phase is dispersed in continuous aqueous phase and forms emulsion, by adjusting containing for PVA
The time of amount, the ratio of oil phase/water phase, and magneton stirring, it is 1.0~1.5 μm to control emulsion diameter；
Step 3, PEG-co-AA microgel cores are synthesized, the emulsion being made is placed in 100w lower 20 minutes of uviol lamp, and ultraviolet light swashs
Luminous initiator produces free radical, and under the principle of radical polymerization, the double bond of PEGDA and AA in oil droplet are opened, and lead
Chain is caused to increase the cross-linking reaction between chain；Therefore, the PEGDA and AA in oil droplet form microgel core, outstanding with 70% ethanol
The method of floating-centrifugation is purified repeatedly for three times, and the method suspend-centrifuged with deionized water removes alcohol；Pass through control
PEGDA and AA ratio 10:0.1~1, the charge density of microgel core is controlled, the minus 6~40mv of potential range is formed；It is made
Protomere core is collected by 16000~50000r/min high speed centrifugations, with the filter membrane of different pore size can selectivity obtain institute
1~the 1000nm of particle size needed, the protomere core being finally collected into is dispersed in 10mlPBS phosphate buffers；
Step 4, PEG-co-AA microgels core surface modification PMAA high charge density shells, microgel core after purification first by
It is distributed in the 0.2mg/mL of polyvinylpyrrolidone, pH 2,0.01M PBS solutions, after 15 minutes, microgel grain is passed through into mistake
Filter, the mode of centrifugation is cleaned, then, and the PVPON micelles treated are dispersed in polymethylacrylic acid PMAA 0.2mg/mL, pH
In 2,0.01M PBS solutions after 15 minutes, clean in aforementioned manners, repeat above method 5~30 times, be formed with hydrogen bond crosslinks
5~30 layers of PVPON/PMAA gel layers；
Step 5, cross-linked stable PMAA gels shell, micro-gel particles are dispersed in 1- ethyls-(3- dimethylaminopropyls) carbon
1.5 hours in acyl diimmonium salt hydrochlorate 5mg/mL and hydroxy thiosuccinimide 5mg/mL 0.01M PBS, PH5 solution；
Afterwards, micelle is separated from solution, and is dipped into 10mg/mL ethylenediamine solution, in carry out between PMAA layers within 14 hours
Covalent cross-linking；Last PEG-co-AA/PMAA composite microgels grain is soaked in pH7.5 0.01M PBS 2 hours, is removed
PVPON and other unreacted reagents, composite microgel grain is suspended in PBS, pH7.4,0.1M after centrifugation-filtering；Should
The size of the composite microgel in the case of different pH4~9 and ionic strength with dynamic light scattering and Zeta electric potential assay method
With powered situation, 100~400 μm of protomere (2) diameter, 0.6~8 μm of altitude range are combined；PMAA microgels layer potential range
Minus 6~0mv, 5~30 layers of the number of plies；
Step 6, the structure on the topological antibacterial structure surface of titanium, deposition of the microgel on titanium surface:Compound protomere is suspended in
In 0.01M, pH7.5 PBS, regulation micelle concentration is 1.0~2.0 × 1010To 1011Individual/ml, the poly L of positively charged is relied
Propylhomoserin positively charged macromolecule layer titanium sheet is dipped into above-mentioned microgel suspension, and due to electrostatic adsorption, microgel will be automatic
Titanium plate surface is adsorbed onto, after 0.5~10 hour, titanium sheet is removed, is alternately rinsed, dried with 0.01M PBS and deionized water,
The titanium sheet prepared is observed under ESEM, confirm microgel absorption, build and screen gel diameter range 100~
400 μm, the antibacterial topological structure surface titanium plate of 0.5~4 μm of 0.6~8 μm of altitude range and gel interstice coverage；
Step 7, beta-alexin -1~3, lactoferrin 1-11LL-37, the similar body of histatins, endogenous antimicrobial are more
Peptide -1 and artificial synthesized cationic antibacterial peptide are loaded into the titanium surface of microgel modification, under the conditions of 36~37 DEG C, and that dries answers
Close microgel modification titanium surface be soaked in concentration be 10~20mL, concentration be 1mg/mL, pH value be 7.4 hBD-3, newborn iron
The similar body of albumen 1-11, LL-37, histatins, protegrins -1 and artificial synthesized cationic antibacterial peptide PBS
In solution, due to powered property on the contrary, hBD-3 is loaded into composite microgel, the load beta-alexin 1 with electrical potential difference is formed
Element -1~3, lactoferrin 1-11, LL-37, the similar body of histatins, protegrins -1 and artificial synthesized sun
Ion antibacterial peptide topology antibacterial structure surface recombination protomere layer, is dried at room temperature, obtains intelligent nano-hydrogel topology knot
Structure antibacterial bone plate.
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| CN101214394A (en) * | 2008-01-10 | 2008-07-09 | 中国人民解放军第三军医大学第三附属医院 | Bone plate with sustained-release antimicrobial peptide |
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