CN104857574A - Intelligent antibacterial nano-hydrogel bone fracture plate with topological structure and manufacturing method thereof - Google Patents
Intelligent antibacterial nano-hydrogel bone fracture plate with topological structure and manufacturing method thereof Download PDFInfo
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Abstract
The invention discloses an intelligent antibacterial nano-hydrogel bone fracture plate with a topological structure. The surface of a substrate is coated with composite micelles, wherein each composite micelle comprises a polyethylene glycol-co-acrylic acid microgel core which serves as inner core and an outer layer controlled release shell which is a pH sensitive polymethylacrylic acid microgel layer; the polyethylene glycol-co-acrylic acid microgel core and the pH sensitive polymethylacrylic acid microgel layer form intelligent charge gradient radial distribution; the composite micelle coating forms a topological antibacterial structure surface on stainless steel, titanium metal or titanium alloy bone fracture plate and is loaded with beta-defensin-1-3, lactoferrin 1-11, LL-37, histadine-rich protein analogues, endogenous antimicrobial peptide-1 and artificially synthesized cationic antimicrobial peptides. The intelligent antibacterial nano-hydrogel bone fracture plate with the topological structure is used as an intelligent simulation response mechanism of contact sterilization when the bacteria are generated on the surface of an orthopedic object and serve as a stimulation factor, so that osteoblasts can quickly grow on the surface of the orthopedic object; the bacterial adhesion is reduced; the bacteria are killed by antimicrobial peptide; target points used for controlling the formation of bacterial biofilms can be really restrained in the initial stage, thereby finally reducing the occurrence rate of orthopedic device related infections.
Description
Technical field
The invention belongs to surgical material and manufacture method thereof, specifically, intelligent nano-hydrogel topological structure antibacterial bone plate.
Background technology
According to international ASIF AO (Arbeit sgem ein schaft fur Osteosynthesesfragen)/ASIF (Association for the Study of Internal Fixation), in open fracture, infection rate is up to about 3 ~ 40%, as used internal fixtion then infection rate increase by more than 30%.Annual in Yue200Wan Li nosocomial infection of U.S. case, about 50% is relevant with implants, not only consumes the huge wealth of society up to 24,000,000,000 dollars, also causes comprising the serious consequences such as even annual 100000 deaths of bacterial resistance, amputation.Trace it to its cause, antibacterial and implant material adhere to the main pathological basis to form complete biomembrane and be " implants infections relating "." implants infections relating " (Orthopedic Device RelatedInfections, ODRI) is one of the most common and serious complication of puzzlement Orthopedic Clinical all the time.For ODRI, generally traditional Therapeutic Method is: on the basis of taking out implants, and infection and necrosis bone and soft tissue and most of antibacterial are removed in thorough debridement; Use the temporary fixed fracture site of exterior fixation bracket; Remaining bacteria eliminated by whole body or the heavy dose of antibiotic of topical application; The technology such as application flap close wound.It is certain that after infecting, row bone grafting, bone are moved or extend and film skeletonization technology recovery bone structure again.
For the traditional treatment technology of ODRI, the cycle is long, cost is large, and many use exterior fixation bracket fix bone, maintain its stability.And less use internal fixation device, with identical with the reason that the implants such as firearm injury light plate screw are then regarded as contraindication at open fracture, only about quantity is 10
3the antibacterial of CFU/ml can adhere to implants and and formed complete biomembrane cause the generation of ODRI, develop and lasting protracted course of disease be basis.And when there is no an implants, even if open fracture local has 10
6cFU/ml antibacterial, also may not cause human body to infect.But extenal fixation has pin site infection rate high (5.9% ~ 19%), not easily fix in setting board of skeleton end fracture, draw point is loosening and have a strong impact on minimal invasive treatment, be not easy to the shortcomings such as nursing.And in setting board of skeleton end fracture, blade plate is the mode of irreplaceable fixing fracture site.Therefore, in the prevention and treatment of ODRI, conventional antibiotic using method: whole body or local application.The former, often cause antibiotic in whole body blood to be difficult to reach effective antimicrobial concentration in damage local because local damage and blood follow destruction; The latter, although the antibiotic that can produce high concentration at local and initial, declines fast subsequently, can not maintain and effectively treat concentration.Therefore, be all difficult to produce effective anti-infectious function to the Fashion and Evolution of ODRI and Traumatic osteomyelitis.Current directed toward bacteria biomembrane, synthesize by suppressing its polysaccharide matrix and promote its decompositions, interference density induction system, add with the surf ace properties of monoclonal antibody and improvement implant to reduce the methods such as the adhesion of antibacterial and material and antibiotic coating, confirm can reduce ODRI incidence rate to a certain extent, but the persistent infection that all can not solve biomembranous formation up hill and dale and cause thus.For this reason, find the method for new change fixture materials surface characteristic or material become current clinical position in the urgent need to, be one of multi-field research focus such as domestic and international microorganism, material, clinical medicine.
Summary of the invention
An object of the present invention is the intelligent nano-hydrogel topological structure antibacterial bone plate providing a kind of powerful infection and anti-bacteria biofilm formation, the compound micelle that this blade plate prepares intelligent charge gradient radial distribution gel particle that PEG-co-AA micelle is core, pH responsive type PMMA microgel layer is controlled release shell.This micelle can not only the abundant antibacterial peptide such as load hBD-3, and because the antibacterial peptides such as hBD-3 in micelle core present positive charge gradient radial distribution, the antibacterial peptides such as hBD-3 are had triple " intellectuality " feature of " contact sterilization " effect and topological antibacterial structure was automatically filled vacancies in the proper order, played to " intellectuality " to low potential nano-hydrogel coating by high potential, to overcome the predicament of conventional crosslinking method and coating facile hydrolysis, load antibacterial peptide amount is few and the half-life is short defect, thus break through the application " bottleneck " that the antibacterial peptides such as hBD-3 prevent and treat ODRI in vivo.
Two of object of the present invention is the manufacture method providing a kind of intelligent nano-hydrogel topological structure antibacterial bone plate.
A kind of intelligent nano-hydrogel topological structure antibacterial bone plate, comprise titanium or titanium alloy blade plate (1), its key is: the cationic antibacterial peptide per unit area concentration about 0.025 ~ 1mg/mL of described titanium or titanium alloy blade plate topology antibacterial structure surface recombination micelle load beta-alexin-1 ~ 3, lactoferrin 1-11, LL-37, histatins analog (histatinanalogue DHVAR-5), protegrins-1 (Protegrins-1) and synthetic.Described titanium or titanium alloy blade plate surface coverage are by Polyethylene Glycol-co-acrylic acid (Poly (ethylene glycol)-co-acrylic acid, PEG-co-AA) microgel core is kernel, outer controlled release shell is pH responsive type polymethylacrylic acid (poly (methacrylic acid, PMAA) the intelligent charge gradient radial distribution compound micelle of microgel layer, this compound micelle coating is at rustless steel, titanium or titanium alloy blade plate form topological antibacterial structure surface recombination micelle load beta-alexin-1 ~ 3, lactoferrin 1-11, LL-37, histatins analog (histatin analogue DHVAR-5), the cationic antibacterial peptide of protegrins-1 (Protegrins-1) and synthetic.Described compound micelle diameter about 100 ~ 400 μm, altitude range 0.6 ~ 8 μm.PEG-co-AA microgel core potential range about negative 6 ~ 40mv, core internal diameter about 1 ~ 1000nm; The about negative 6 ~ 0mv of PMAA microgel layer potential range, the number of plies about 5 ~ 30 layers.This compound micelle possesses the characteristic that electric charge shifts to low potential automatically from high potential.Described rustless steel, titanium or titanium alloy blade plate topology antibacterial structure surface recombination micelle (3) spacing range about 0.5 ~ 4 μm.
A manufacture method for intelligent nano-hydrogel topological structure antibacterial bone plate, its key is, carries out as follows:
Step 1, titanium or (1) surface preparation of titanium alloy blade plate and the electric treatment of titanium surface band.Titanium or titanium alloy blade plate (1) carry out hydrophilic treated each 10 ~ 20 minutes with distilled water, acetone, 75% ethanol, distilled water ultrasonic cleaning, panel successively, insert 0.2mg/mL, mean molecule quantity is 30 ~ 50k, many PLL (poly-l-lysine of pH9, PLL) 2 ~ 3 hours, form positively charged macromolecule layer at titanium alloy substrate, carry out initial layer deposition, clean with deionized water after taking-up, at room temperature dried for standby.
Step 2, preparation PEGDA/AA emulsion (2).Be averaged polyethylene glycol acrylate (the Polyethylene Glycol Diacrylate of molecular weight 500 ~ 600 respectively, PEGDA) 200 μ l, acrylic monomers (acrylic acid, AA) after 50 DEG C of distillations, 0 ~ 20 μ l is left and taken, and light trigger (Darocur 1173, Ciba) 10 μ l, are jointly dissolved in dichloromethane (dichloromethane, DCM) 1ml and form oil phase.1 ~ 10% polyvinyl alcohol (PVA) 1 ~ 3ml, as emulsifying agent, is dissolved in 10ml deionized water and forms aqueous phase.In the stirring 10 ~ 20 minutes of high speed magneton (1000rpm), oil phase is dispersed in continuous print aqueous phase and forms emulsion.By adjusting the content of PVA, the ratio of oil phase/water phase, and the time that magneton stirs, control emulsion diameter about 1.0 ~ 1.5 μm.
Step 3, synthesis PEG-co-AA microgel core (2).The emulsion made is placed in lower 20 minutes of the uviol lamp of 100w.Ultraviolet excitation light trigger produces free radical, and under the principle of radical polymerization, the double bond of PEGDA and AA in oil droplet is opened, and causes cross-linking reaction between chain growth and chain.Therefore, PEGDA and AA in oil droplet defines microgel core, repeatedly carries out purification three times with 70% ethanol suspension-centrifugal method, removes ethanol by deionized water suspension-centrifugal method.By ratio 10:0.1 ~ 1 of control PEGDA and AA, control the charge density of microgel core, form electromotive force and be about the about negative 6 ~ 40mv of scope.The micelle core made is collected by 16000 ~ 50000r/min high speed centrifugation, and optionally can obtain required size 1 ~ 1000nm with the filter membrane of different pore size, the micelle core finally collected is dispersed in 10mlPBS phosphate buffer.
Step 4, PEG-co-AA microgel core finishing PMAA high charge density shell (2).First microgel core after purification be dispersed to polyvinylpyrrolidone (poly (N-vinylpyrrolidone), PVPON) (0.2mg/mL in PBS solution, pH 2,0.01M PBS), after 15 minutes, by microgel grain by filtering, centrifugal mode is cleaned.Subsequently, the micelle of PVPON process to be dispersed in the PBS solution of polymethylacrylic acid PMAA (0.2mg/mL, pH 2,0.01M PBS), after 15 minutes, to clean with said method.Repeat above method 5 ~ 30 times, be formed with 5 ~ 30 layers of PVPON/PMAA gel layer of hydrogen bond crosslinks.
Step 5, cross-linked stable PMAA gel shell (2).Micro-gel particles to be dispersed in the 0.01M PBS solution (PH5) of 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate 5mg/mL and hydroxy thiosuccinimide 5mg/mL 1.5 hours; Afterwards, micelle is separated from solution, and is dipped in ethylenediamine (ethylenediamine, the EDA) solution (pH5.8) of 10mg/mL the covalent cross-linking carried out for 14 hours between PMAA layer; Finally, in order to remove PVPON and other reagent unreacted, PEG-co-AA/PMAA composite microgel grain to be soaked in the 0.01M PBS of pH7.5 2 hours, centrifugal-filter after composite microgel grain be suspended in (pH7.4,0.1M) in PBS.The size of application dynamic light scattering and Zeta electric potential assay method composite microgel when different pH4 ~ 9 and ionic strength and charged situation.Compound micelle (2) diameter about 100 ~ 400 μm, altitude range 0.6 ~ 8 μm.The about negative 6 ~ 0mv of PMAA microgel layer potential range, the number of plies about 5 ~ 30 layers.This compound micelle possesses the characteristic that electric charge shifts to low potential automatically from high potential.
Step 6, the structure (3) on titanium topology antibacterial structure surface.Microgel is in the deposition on titanium surface: compound micelle is suspended in the PBS of 0.01M, pH7.5, regulates micelle concentration to be 1.0 ~ 2.0 × 10
10to 10
11individual/ml.Many PLLs of positively charged positively charged macromolecule layer titanium sheet is dipped in above-mentioned microgel suspension, due to electrostatic adsorption, microgel by automatic absorbing to titanium plate surface.After 0.5 ~ 10 hour, titanium sheet is removed, and alternately rinses with 0.01M PBS and deionized water, dry.The titanium sheet prepared is observed under scanning electron microscope, confirms the absorption of microgel.Build and screen the antibacterial topological structure surface titanium plate of gel diameter range 100 ~ 400 μm, altitude range 0.6 ~ 8 μm and gel interstice coverage 0.5 ~ 4 μm.
Step 7, the cationic antibacterial peptide of beta-alexin-1 ~ 3, lactoferrin 1-11, LL-37, histatins analog (histatin analogue DHVAR-5), protegrins-1 (Protegrins-1) and synthetic is loaded into the titanium surface (4) that microgel is modified.Under 36 ~ 37 DEG C of conditions, the titanium surface that dry composite microgel is modified is soaked in that concentration is 10 ~ 20mL, concentration is 1mg/mL, pH value be 7.4 hBD-3 or lactoferrin 1-11PBS solution in, because charged character is contrary, hBD-3 is loaded in composite microgel.Form cationic antibacterial peptide topology antibacterial structure surface recombination micelle layer (4) carrying beta-alexin-1 ~ 3, lactoferrin 1-11, LL-37, histatins analog (histatin analogue DHVAR-5), protegrins-1 (Protegrins-1) and synthetic with electric potential difference, dry under room temperature, obtain intelligent nano-hydrogel topological structure antibacterial bone plate.
The present invention's intelligent nano-hydrogel topological structure antibacterial bone plate (New Zealand white rabbit) and external medicine kinetic measurement in body confirm, constructing diameter is that the PEG-co-AA micelle of 200 ~ 300nm is with loading cation antibacterial peptide, make it to play when antibacterial is present in implants surface, it is as the intelligent stimuli responsive mechanism of stimulating factor " contact sterilization " (contact-killing), there is the effect of fixing fracture site and powerful infection and anti-bacteria biofilm formation, effective antimicrobial concentration of 10 days can be maintained in local.
Beneficial effect: the emulsion polymerization technology in Applied Surface Chemistry technology of the present invention, multilayer polymeric technology, electrostatic deposition techniques prepare intelligent nano-hydrogel topological structure antibacterial bone plate, not only breach short " bottleneck " of half-life in antibacterial peptide aqueous solution, key makes it to play when antibacterial is present in implants surface, and it is as the intelligent stimuli responsive mechanism of stimulating factor " contact sterilization " (contact-killing).Otherwise, as antibacterial do not exist time, then retain antibacterial peptide in blade plate surface.And changing surface topography and the chemical constitution of biomaterial simultaneously, its three-dimensional topology surface is very beneficial for osteoblastic tactophily and propagation.Thus be embodied as the growth and reduce bacterial adhesion and utilize antibacterial peptide kill bacteria fast on implants surface such as osteocyte, cause and really " target spot " of biomembranous for anti-bacteria formation is controlled at its initial period, finally to reduce the incidence rate of " implants infections relating ".Compare with other slow release methods, this technology has and is easy to (1) and quickly and easily builds surface topology by the electrostatic adsorption between microgel and substrate; (2) kept the structure and fuction of the small-molecular peptides such as antibacterial peptide or albumen to stablize by ionic bond; (3) by controlling metal material surface roughness and micelle spacing, be beneficial to bone or fibroblast growth propagation and " intellectuality " advantage of anti-bacteria tactophily and biofilm formation, meet the development trend of " the third tactful " and " smart material " of current control " fixture infections relating ".The biomembranous Fashion and Evolution of direct suppression, obtains the curative effect of control ODRI.Break through traditionally taboo, fracture caused by ODRI and open or firearm injury is carried out to generation, the development of I phase reparation and control Traumatic osteomyelitis, thus reach Shorten the Treatment Process, significantly improve the object of curative effect.The present invention not only has the feature of fixing fracture site and powerful infection and anti-bacteria biofilm formation, and possess " intellectuality " just play the intelligent stimuli responsive mechanism of " contact sterilization " by automatically to distribute to low potential region antibacterial peptide and antibacterial of high potential region as stimulating factor.
Accompanying drawing explanation
Fig. 1 is preparation flow and the effect schematic diagram of blade plate of the present invention;
Detailed description of the invention
Embodiment 1
The manufacture method of intelligent nano-hydrogel topological structure antibacterial bone plate, carry out as follows:
Step 1, titanium or (1) surface preparation of titanium alloy blade plate and the electric treatment of titanium surface band.Titanium or titanium alloy blade plate (1) carry out hydrophilic treated each 10 ~ 20 minutes with distilled water, acetone, 75% ethanol, distilled water ultrasonic cleaning, panel successively, insert 0.2mg/mL, mean molecule quantity is 30 ~ 50k, many PLL (poly-l-lysine of pH 9, PLL) 2 ~ 3 hours, form positively charged macromolecule layer at titanium alloy substrate, carry out initial layer deposition, clean with deionized water after taking-up, at room temperature dried for standby.
Step 2, preparation PEGDA/AA emulsion.Be averaged polyethylene glycol acrylate (the Polyethylene Glycol Diacrylate of molecular weight 500 ~ 600 respectively, PEGDA) 200 μ l, acrylic monomers (acrylicacid, AA) after 50 DEG C of distillations, 0 ~ 20 μ l is left and taken, and light trigger (Darocur 1173, Ciba) 10 μ l, are jointly dissolved in dichloromethane (dichloromethane, DCM) 1ml and form oil phase.1 ~ 10% polyvinyl alcohol (PVA) 1 ~ 3ml, as emulsifying agent, is dissolved in 10ml deionized water and forms aqueous phase.In the stirring 10 ~ 20 minutes of high speed magneton (1000rpm), oil phase is dispersed in continuous print aqueous phase and forms emulsion.By adjusting the content of PVA, the ratio of oil phase/water phase, and the time that magneton stirs, control emulsion diameter about 1.0 ~ 1.5 μm.
Step 3, synthesis PEG-co-AA microgel core.The emulsion made is placed in lower 20 minutes of the uviol lamp of 100w.Ultraviolet excitation light trigger produces free radical, and under the principle of radical polymerization, the double bond of PEGDA and AA in oil droplet is opened, and causes cross-linking reaction between chain growth and chain.Therefore, PEGDA and AA in oil droplet defines microgel core, repeatedly carries out purification three times with 70% ethanol suspension-centrifugal method, removes ethanol by deionized water suspension-centrifugal method.By ratio 10:0.1 ~ 1 of control PEGDA and AA, control the charge density of microgel core, form electromotive force and be about the about negative 6 ~ 40mv of scope.The micelle core made is collected by 16000 ~ 50000r/min high speed centrifugation, and optionally can obtain required size 1 ~ 1000nm with the filter membrane of different pore size, the micelle core finally collected is dispersed in 10mlPBS phosphate buffer.
Step 4, PEG-co-AA microgel core finishing PMAA high charge density shell.First microgel core after purification be dispersed to polyvinylpyrrolidone (poly (N-vinylpyrrolidone), PVPON) (0.2mg/mL in PBS solution, pH 2,0.01M PBS), after 15 minutes, by microgel grain by filtering, centrifugal mode is cleaned.Subsequently, the micelle of PVPON process to be dispersed in the PBS solution of polymethylacrylic acid PMAA (0.2mg/mL, pH 2,0.01M PBS), after 15 minutes, to clean with said method.Repeat above method 5 ~ 30 times, be formed with 5 ~ 30 layers of PVPON/PMAA gel layer of hydrogen bond crosslinks.
Step 5, cross-linked stable PMAA gel shell.Micro-gel particles to be dispersed in the 0.01MPBS solution (PH5) of 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate 5mg/mL and hydroxy thiosuccinimide 5mg/mL 1.5 hours; Afterwards, micelle is separated from solution, and is dipped in ethylenediamine (ethylenediamine, the EDA) solution (pH5.8) of 10mg/mL the covalent cross-linking carried out for 14 hours between PMAA layer; Finally, in order to remove PVPON and other reagent unreacted, PEG-co-AA/PMAA composite microgel grain to be soaked in the 0.01M PBS of pH7.5 2 hours, centrifugal-filter after composite microgel grain be suspended in (pH7.4,0.1M) in PBS.The size of application dynamic light scattering and Zeta electric potential assay method composite microgel when different pH4 ~ 9 and ionic strength and charged situation.Compound micelle 2 diameter about 100 ~ 400 μm, altitude range 0.6 ~ 8 μm.The about negative 6 ~ 0mv of PMAA microgel layer potential range, the number of plies about 5 ~ 30 layers.This compound micelle 2 possesses the characteristic that electric charge shifts to low potential automatically from high potential.
Step 6, the structure on titanium topology antibacterial structure surface.Microgel is in the deposition on titanium surface: compound micelle 2 is suspended in the PBS of 0.01M, pH7.5, regulates micelle concentration to be 1.0 ~ 2.0 × 10
10to 10
11individual/ml.Many PLLs of positively charged positively charged macromolecule layer titanium sheet is dipped in above-mentioned microgel suspension, due to electrostatic adsorption, microgel by automatic absorbing to titanium plate surface.After 0.5 ~ 10 hour, titanium sheet is removed, and alternately rinses with 0.01M PBS and deionized water, dry.The titanium sheet prepared is observed under scanning electron microscope, confirms the absorption of microgel.Build and screen the antibacterial topological structure surface titanium plate of gel diameter range 100 ~ 400 μm, altitude range 0.6 ~ 8 μm and gel interstice coverage 0.5 ~ 4 μm.
Step 7, beta-alexin-1 ~ 3 is loaded into the titanium surface that microgel is modified.Under 36 ~ 37 DEG C of conditions, the titanium surface that dry composite microgel is modified is soaked in that concentration is 10 ~ 20mL, concentration is 1mg/mL, pH value is in hBD-1 ~ 3PBS solution of 7.4, and because charged character is contrary, hBD-1 ~ 3 are loaded in composite microgel.Form year beta-alexin-1 ~ 3 topological antibacterial structure surface recombination micelle layer with electric potential difference, dry under room temperature, obtain intelligent nano-hydrogel topological structure antibacterial bone plate.
Step 8, antibacterial peptide slow release steel plate step 7 obtained is placed in 40 ° of incubators, solidifies after 10 minutes, encapsulation, oxirane disinfection.
Finally obtain as shown in Figure 1, intelligent nano-hydrogel topological structure antibacterial bone plate, titanium described in described titanium blade plate or titanium alloy blade plate topology antibacterial structure surface recombination micelle 2 load beta-alexin-1 ~ 3 per unit area concentration about 0.025 ~ 1mg/mL.Described titanium or titanium alloy blade plate surface coverage are kernel by PEG-co-AA microgel core, outer controlled release shell is the intelligent charge gradient radial distribution compound micelle 2 of pH responsive type polymethylacrylic acid PMAA microgel layer, this compound micelle 2 coating diameter about 100 ~ 400 μm, altitude range 0.6 ~ 8 μm.PEG-co-AA microgel core potential range about negative 6 ~ 40mv, core internal diameter about 1 ~ 1000nm; The about negative 6 ~ 0mv of PMAA microgel layer potential range, the number of plies about 5 ~ 30 layers.This compound micelle 2 possesses the characteristic that electric charge shifts to low potential automatically from high potential.Described titanium or titanium alloy blade plate topology antibacterial structure surface recombination micelle 2 spacing range about 0.5 ~ 4 μm.
Embodiment 2
The manufacture method of intelligent nano-hydrogel topological structure antibacterial bone plate, carry out as follows:
Step 1, titanium or (1) surface preparation of titanium alloy blade plate and the electric treatment of titanium surface band.Titanium or titanium alloy blade plate (1) carry out hydrophilic treated each 10 ~ 20 minutes with distilled water, acetone, 75% ethanol, distilled water ultrasonic cleaning, panel successively, insert 0.2mg/mL, mean molecule quantity is 30 ~ 50k, many PLL (poly-l-lysine of pH 9, PLL) 2 ~ 3 hours, form positively charged macromolecule layer at titanium alloy substrate, carry out initial layer deposition, clean with deionized water after taking-up, at room temperature dried for standby.
Step 2, preparation PEGDA/AA emulsion.Be averaged polyethylene glycol acrylate (the Polyethylene Glycol Diacrylate of molecular weight 500 ~ 600 respectively, PEGDA) 200 μ l, acrylic monomers (acrylicacid, AA) after 50 DEG C of distillations, 0 ~ 20 μ l is left and taken, and light trigger (Darocur 1173, Ciba) 10 μ l, are jointly dissolved in dichloromethane (dichloromethane, DCM) 1ml and form oil phase.1 ~ 10% polyvinyl alcohol (PVA) 1 ~ 3ml, as emulsifying agent, is dissolved in 10ml deionized water and forms aqueous phase.In the stirring 10 ~ 20 minutes of high speed magneton (1000rpm), oil phase is dispersed in continuous print aqueous phase and forms emulsion.By adjusting the content of PVA, the ratio of oil phase/water phase, and the time that magneton stirs, control emulsion diameter about 1.0 ~ 1.5 μm.
Step 3, synthesis PEG-co-AA microgel core.The emulsion made is placed in lower 20 minutes of the uviol lamp of 100w.Ultraviolet excitation light trigger produces free radical, and under the principle of radical polymerization, the double bond of PEGDA and AA in oil droplet is opened, and causes cross-linking reaction between chain growth and chain.Therefore, PEGDA and AA in oil droplet defines microgel core, repeatedly carries out purification three times with 70% ethanol suspension-centrifugal method, removes ethanol by deionized water suspension-centrifugal method.By ratio 10:0.1 ~ 1 of control PEGDA and AA, control the charge density of microgel core, form electromotive force and be about the about negative 6 ~ 40mv of scope.The micelle core made is collected by 16000 ~ 50000r/min high speed centrifugation, and optionally can obtain required size 1 ~ 1000nm with the filter membrane of different pore size, the micelle core finally collected is dispersed in 10mlPBS phosphate buffer.
Step 4, PEG-co-AA microgel core finishing PMAA high charge density shell.First microgel core after purification be dispersed to polyvinylpyrrolidone (poly (N-vinylpyrrolidone), PVPON) (0.2mg/mL in PBS solution, pH 2,0.01M PBS), after 15 minutes, by microgel grain by filtering, centrifugal mode is cleaned.Subsequently, the micelle of PVPON process to be dispersed in the PBS solution of polymethylacrylic acid PMAA (0.2mg/mL, pH 2,0.01M PBS), after 15 minutes, to clean with said method.Repeat above method 5 ~ 30 times, be formed with 5 ~ 30 layers of PVPON/PMAA gel layer of hydrogen bond crosslinks.
Step 5, cross-linked stable PMAA gel shell.Micro-gel particles to be dispersed in the 0.01MPBS solution (PH5) of 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate 5mg/mL and hydroxy thiosuccinimide 5mg/mL 1.5 hours; Afterwards, micelle is separated from solution, and is dipped in ethylenediamine (ethylenediamine, the EDA) solution (pH5.8) of 10mg/mL the covalent cross-linking carried out for 14 hours between PMAA layer; Finally, in order to remove PVPON and other reagent unreacted, PEG-co-AA/PMAA composite microgel grain to be soaked in the 0.01M PBS of pH7.5 2 hours, centrifugal-filter after composite microgel grain be suspended in (pH7.4,0.1M) in PBS.The size of application dynamic light scattering and Zeta electric potential assay method composite microgel when different pH4 ~ 9 and ionic strength and charged situation.Compound micelle 2 diameter about 100 ~ 400 μm, altitude range 0.6 ~ 8 μm.The about negative 6 ~ 0mv of PMAA microgel layer potential range, the number of plies about 5 ~ 30 layers.This compound micelle 2 possesses the characteristic that electric charge shifts to low potential automatically from high potential.
Step 6, the structure on titanium topology antibacterial structure surface.Microgel is in the deposition on titanium surface: compound micelle 2 is suspended in the PBS of 0.01M, pH7.5, regulates micelle concentration to be 1.0 ~ 2.0 × 10
10to 10
11individual/ml.Many PLLs of positively charged positively charged macromolecule layer titanium sheet is dipped in above-mentioned microgel suspension, due to electrostatic adsorption, microgel by automatic absorbing to titanium plate surface.After 0.5 ~ 10 hour, titanium sheet is removed, and alternately rinses with 0.01M PBS and deionized water, dry.The titanium sheet prepared is observed under scanning electron microscope, confirms the absorption of microgel.Build and screen the antibacterial topological structure surface titanium plate of gel diameter range 100 ~ 400 μm, altitude range 0.6 ~ 8 μm and gel interstice coverage 0.5 ~ 4 μm.
Step 7, LL-37 is loaded into the titanium surface that microgel is modified.Under 36 ~ 37 DEG C of conditions, the titanium surface that dry composite microgel is modified is soaked in that concentration is 10 ~ 20mL, concentration is 1mg/mL, pH value is in the LL-37PBS solution of 7.4, and because charged character is contrary, LL-37 is loaded in composite microgel.Form the LL-37 topology antibacterial structure surface recombination micelle layer with electric potential difference, dry under room temperature, obtain intelligent nano-hydrogel topological structure antibacterial bone plate.
Step 8, antibacterial peptide slow release steel plate step 7 obtained is placed in 40 ° of incubators, solidifies after 10 minutes, encapsulation, oxirane disinfection.
Finally obtain as shown in Figure 1, intelligent nano-hydrogel topological structure antibacterial bone plate, titanium described in described titanium blade plate or titanium alloy blade plate topology antibacterial structure surface recombination micelle 2 load LL-37 per unit area concentration about 0.025 ~ 1mg/mL.Described titanium or titanium alloy blade plate surface coverage are kernel by PEG-co-AA microgel core, outer controlled release shell is the intelligent charge gradient radial distribution compound micelle 2 of pH responsive type polymethylacrylic acid PMAA microgel layer, this compound micelle 2 coating diameter about 100 ~ 400 μm, altitude range 0.6 ~ 8 μm.PEG-co-AA microgel core potential range about negative 6 ~ 40mv, core internal diameter about 1 ~ 1000nm; The about negative 6 ~ 0mv of PMAA microgel layer potential range, the number of plies about 5 ~ 30 layers.This compound micelle 2 possesses the characteristic that electric charge shifts to low potential automatically from high potential.Described titanium or titanium alloy blade plate topology antibacterial structure surface recombination micelle 2 spacing range about 0.5 ~ 4 μm.
Embodiment 3
The manufacture method of intelligent nano-hydrogel topological structure antibacterial bone plate, carry out as follows:
Step 1, titanium or (1) surface preparation of titanium alloy blade plate and the electric treatment of titanium surface band.Titanium or titanium alloy blade plate (1) carry out hydrophilic treated each 10 ~ 20 minutes with distilled water, acetone, 75% ethanol, distilled water ultrasonic cleaning, panel successively, insert 0.2mg/mL, mean molecule quantity is 30 ~ 50k, many PLL (poly-l-lysine of pH 9, PLL) 2 ~ 3 hours, form positively charged macromolecule layer at titanium alloy substrate, carry out initial layer deposition, clean with deionized water after taking-up, at room temperature dried for standby.
Step 2, preparation PEGDA/AA emulsion.Be averaged polyethylene glycol acrylate (the Polyethylene Glycol Diacrylate of molecular weight 500 ~ 600 respectively, PEGDA) 200 μ l, acrylic monomers (acrylicacid, AA) after 50 DEG C of distillations, 0 ~ 20 μ l is left and taken, and light trigger (Darocur 1173, Ciba) 10 μ l, are jointly dissolved in dichloromethane (dichloromethane, DCM) 1ml and form oil phase.1 ~ 10% polyvinyl alcohol (PVA) 1 ~ 3ml, as emulsifying agent, is dissolved in 10ml deionized water and forms aqueous phase.In the stirring 10 ~ 20 minutes of high speed magneton (1000rpm), oil phase is dispersed in continuous print aqueous phase and forms emulsion.By adjusting the content of PVA, the ratio of oil phase/water phase, and the time that magneton stirs, control emulsion diameter about 1.0 ~ 1.5 μm.
Step 3, synthesis PEG-co-AA microgel core.The emulsion made is placed in lower 20 minutes of the uviol lamp of 100w.Ultraviolet excitation light trigger produces free radical, and under the principle of radical polymerization, the double bond of PEGDA and AA in oil droplet is opened, and causes cross-linking reaction between chain growth and chain.Therefore, PEGDA and AA in oil droplet defines microgel core, repeatedly carries out purification three times with 70% ethanol suspension-centrifugal method, removes ethanol by deionized water suspension-centrifugal method.By ratio 10:0.1 ~ 1 of control PEGDA and AA, control the charge density of microgel core, form electromotive force and be about the about negative 6 ~ 40mv of scope.The micelle core made is collected by 16000 ~ 50000r/min high speed centrifugation, and optionally can obtain required size 1 ~ 1000nm with the filter membrane of different pore size, the micelle core finally collected is dispersed in 10mlPBS phosphate buffer.
Step 4, PEG-co-AA microgel core finishing PMAA high charge density shell.First microgel core after purification be dispersed to polyvinylpyrrolidone (poly (N-vinylpyrrolidone), PVPON) (0.2mg/mL in PBS solution, pH 2,0.01M PBS), after 15 minutes, by microgel grain by filtering, centrifugal mode is cleaned.Subsequently, the micelle of PVPON process to be dispersed in the PBS solution of polymethylacrylic acid PMAA (0.2mg/mL, pH 2,0.01M PBS), after 15 minutes, to clean with said method.Repeat above method 5 ~ 30 times, be formed with 5 ~ 30 layers of PVPON/PMAA gel layer of hydrogen bond crosslinks.
Step 5, cross-linked stable PMAA gel shell.Micro-gel particles to be dispersed in the 0.01MPBS solution (PH5) of 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate 5mg/mL and hydroxy thiosuccinimide 5mg/mL 1.5 hours; Afterwards, micelle is separated from solution, and is dipped in ethylenediamine (ethylenediamine, the EDA) solution (pH5.8) of 10mg/mL the covalent cross-linking carried out for 14 hours between PMAA layer; Finally, in order to remove PVPON and other reagent unreacted, PEG-co-AA/PMAA composite microgel grain to be soaked in the 0.01M PBS of pH7.5 2 hours, centrifugal-filter after composite microgel grain be suspended in (pH7.4,0.1M) in PBS.The size of application dynamic light scattering and Zeta electric potential assay method composite microgel when different pH4 ~ 9 and ionic strength and charged situation.Compound micelle 2 diameter about 100 ~ 400 μm, altitude range 0.6 ~ 8 μm.The about negative 6 ~ 0mv of PMAA microgel layer potential range, the number of plies about 5 ~ 30 layers.This compound micelle 2 possesses the characteristic that electric charge shifts to low potential automatically from high potential.
Step 6, the structure on titanium topology antibacterial structure surface.Microgel is in the deposition on titanium surface: compound micelle 2 is suspended in the PBS of 0.01M, pH7.5, regulates micelle concentration to be 1.0 ~ 2.0 × 10
10to 10
11individual/ml.Many PLLs of positively charged positively charged macromolecule layer titanium sheet is dipped in above-mentioned microgel suspension, due to electrostatic adsorption, microgel by automatic absorbing to titanium plate surface.After 0.5 ~ 10 hour, titanium sheet is removed, and alternately rinses with 0.01M PBS and deionized water, dry.The titanium sheet prepared is observed under scanning electron microscope, confirms the absorption of microgel.Build and screen the antibacterial topological structure surface titanium plate of gel diameter range 100 ~ 400 μm, altitude range 0.6 ~ 8 μm and gel interstice coverage 0.5 ~ 4 μm.
Step 7, histatins analog (histatin analogue DHVAR-5) is loaded into the titanium surface that microgel is modified.Under 36 ~ 37 DEG C of conditions, the titanium surface that dry composite microgel is modified is soaked in that concentration is 10 ~ 20mL, concentration is 1mg/mL, pH value is in the hBD-3PBS solution of 7.4, because charged character is contrary, histatins analog (histatin analogue DHVAR-5) is loaded in composite microgel.Form the topological antibacterial structure surface recombination micelle layer of histatins analog (histatin analogue DHVAR-5) with electric potential difference, dry under room temperature, obtain intelligent nano-hydrogel topological structure antibacterial bone plate.
Step 8, antibacterial peptide slow release steel plate step 7 obtained is placed in 40 ° of incubators, solidifies after 10 minutes, encapsulation, oxirane disinfection.
Finally obtain as shown in Figure 1, intelligent nano-hydrogel topological structure antibacterial bone plate, titanium described in described titanium blade plate or titanium alloy blade plate topology antibacterial structure surface recombination micelle 2 load beta-alexin-3 per unit area concentration about 0.025 ~ 1mg/mL.Described titanium or titanium alloy blade plate surface coverage are kernel by PEG-co-AA microgel core, outer controlled release shell is the intelligent charge gradient radial distribution compound micelle 2 of pH responsive type polymethylacrylic acid PMAA microgel layer, this compound micelle 2 coating diameter about 100 ~ 400 μm, altitude range 0.6 ~ 8 μm.PEG-co-AA microgel core potential range about negative 6 ~ 40mv, core internal diameter about 1 ~ 1000nm; The about negative 6 ~ 0mv of PMAA microgel layer potential range, the number of plies about 5 ~ 30 layers.This compound micelle 2 possesses the characteristic that electric charge shifts to low potential automatically from high potential.Described titanium or titanium alloy blade plate topology antibacterial structure surface recombination micelle 2 spacing range about 0.5 ~ 4 μm.
Embodiment 4
The manufacture method of intelligent nano-hydrogel topological structure antibacterial bone plate, carry out as follows:
Step 1, titanium or (1) surface preparation of titanium alloy blade plate and the electric treatment of titanium surface band.Titanium or titanium alloy blade plate (1) carry out hydrophilic treated each 10 ~ 20 minutes with distilled water, acetone, 75% ethanol, distilled water ultrasonic cleaning, panel successively, insert 0.2mg/mL, mean molecule quantity is 30 ~ 50k, many PLL (poly-l-lysine of pH 9, PLL) 2 ~ 3 hours, form positively charged macromolecule layer at titanium alloy substrate, carry out initial layer deposition, clean with deionized water after taking-up, at room temperature dried for standby.
Step 2, preparation PEGDA/AA emulsion.Be averaged polyethylene glycol acrylate (the Polyethylene Glycol Diacrylate of molecular weight 500 ~ 600 respectively, PEGDA) 200 μ l, acrylic monomers (acrylicacid, AA) after 50 DEG C of distillations, 0 ~ 20 μ l is left and taken, and light trigger (Darocur 1173, Ciba) 10 μ l, are jointly dissolved in dichloromethane (dichloromethane, DCM) 1ml and form oil phase.1 ~ 10% polyvinyl alcohol (PVA) 1 ~ 3ml, as emulsifying agent, is dissolved in 10ml deionized water and forms aqueous phase.In the stirring 10 ~ 20 minutes of high speed magneton (1000rpm), oil phase is dispersed in continuous print aqueous phase and forms emulsion.By adjusting the content of PVA, the ratio of oil phase/water phase, and the time that magneton stirs, control emulsion diameter about 1.0 ~ 1.5 μm.
Step 3, synthesis PEG-co-AA microgel core.The emulsion made is placed in lower 20 minutes of the uviol lamp of 100w.Ultraviolet excitation light trigger produces free radical, and under the principle of radical polymerization, the double bond of PEGDA and AA in oil droplet is opened, and causes cross-linking reaction between chain growth and chain.Therefore, PEGDA and AA in oil droplet defines microgel core, repeatedly carries out purification three times with 70% ethanol suspension-centrifugal method, removes ethanol by deionized water suspension-centrifugal method.By ratio 10:0.1 ~ 1 of control PEGDA and AA, control the charge density of microgel core, form electromotive force and be about the about negative 6 ~ 40mv of scope.The micelle core made is collected by 16000 ~ 50000r/min high speed centrifugation, and optionally can obtain required size 1 ~ 1000nm with the filter membrane of different pore size, the micelle core finally collected is dispersed in 10mlPBS phosphate buffer.
Step 4, PEG-co-AA microgel core finishing PMAA high charge density shell.First microgel core after purification be dispersed to polyvinylpyrrolidone (poly (N-vinylpyrrolidone), PVPON) (0.2mg/mL in PBS solution, pH 2,0.01M PBS), after 15 minutes, by microgel grain by filtering, centrifugal mode is cleaned.Subsequently, the micelle of PVPON process to be dispersed in the PBS solution of polymethylacrylic acid PMAA (0.2mg/mL, pH 2,0.01M PBS), after 15 minutes, to clean with said method.Repeat above method 5 ~ 30 times, be formed with 5 ~ 30 layers of PVPON/PMAA gel layer of hydrogen bond crosslinks.
Step 5, cross-linked stable PMAA gel shell.Micro-gel particles to be dispersed in the 0.01MPBS solution (PH5) of 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate 5mg/mL and hydroxy thiosuccinimide 5mg/mL 1.5 hours; Afterwards, micelle is separated from solution, and is dipped in ethylenediamine (ethylenediamine, the EDA) solution (pH5.8) of 10mg/mL the covalent cross-linking carried out for 14 hours between PMAA layer; Finally, in order to remove PVPON and other reagent unreacted, PEG-co-AA/PMAA composite microgel grain to be soaked in the 0.01M PBS of pH7.5 2 hours, centrifugal-filter after composite microgel grain be suspended in (pH7.4,0.1M) in PBS.The size of application dynamic light scattering and Zeta electric potential assay method composite microgel when different pH4 ~ 9 and ionic strength and charged situation.Compound micelle 2 diameter about 100 ~ 400 μm, altitude range 0.6 ~ 8 μm.The about negative 6 ~ 0mv of PMAA microgel layer potential range, the number of plies about 5 ~ 30 layers.This compound micelle 2 possesses the characteristic that electric charge shifts to low potential automatically from high potential.
Step 6, the structure on titanium topology antibacterial structure surface.Microgel is in the deposition on titanium surface: compound micelle 2 is suspended in the PBS of 0.01M, pH7.5, regulates micelle concentration to be 1.0 ~ 2.0 × 10
10to 10
11individual/ml.Many PLLs of positively charged positively charged macromolecule layer titanium sheet is dipped in above-mentioned microgel suspension, due to electrostatic adsorption, microgel by automatic absorbing to titanium plate surface.After 0.5 ~ 10 hour, titanium sheet is removed, and alternately rinses with 0.01M PBS and deionized water, dry.The titanium sheet prepared is observed under scanning electron microscope, confirms the absorption of microgel.Build and screen the antibacterial topological structure surface titanium plate of gel diameter range 100 ~ 400 μm, altitude range 0.6 ~ 8 μm and gel interstice coverage 0.5 ~ 4 μm.
Step 7, beta-alexin-1 is loaded into the titanium surface that microgel is modified.Under 36 ~ 37 DEG C of conditions, the titanium surface that dry composite microgel is modified is soaked in that concentration is 10 ~ 20mL, concentration is 1mg/mL, pH value is in the lactoferrin 1-11PBS solution of 7.4, because charged character is contrary, lactoferrin 1-11 is loaded in composite microgel.Form the lactoferrin 1-11 topology antibacterial structure surface recombination micelle layer with electric potential difference, dry under room temperature, obtain intelligent nano-hydrogel topological structure antibacterial bone plate.
Step 8, antibacterial peptide slow release steel plate step 7 obtained is placed in 40 ° of incubators, solidifies after 10 minutes, encapsulation, oxirane disinfection.
Finally obtain as shown in Figure 1, intelligent nano-hydrogel topological structure antibacterial bone plate, titanium described in described titanium blade plate or titanium alloy blade plate topology antibacterial structure surface recombination micelle 2 load lactoferrin 1-11 per unit area concentration about 0.025 ~ 1mg/mL.Described titanium or titanium alloy blade plate surface coverage are kernel by PEG-co-AA microgel core, outer controlled release shell is the intelligent charge gradient radial distribution compound micelle 2 of pH responsive type polymethylacrylic acid PMAA microgel layer, this compound micelle 2 coating diameter about 100 ~ 400 μm, altitude range 0.6 ~ 8 μm.PEG-co-AA microgel core potential range about negative 6 ~ 40mv, core internal diameter about 1 ~ 1000nm; The about negative 6 ~ 0mv of PMAA microgel layer potential range, the number of plies about 5 ~ 30 layers.This compound micelle 2 possesses the characteristic that electric charge shifts to low potential automatically from high potential.Described titanium or titanium alloy blade plate topology antibacterial structure surface recombination micelle spacing range about 0.5 ~ 4 μm.
Claims (7)
1. an intelligent nano-hydrogel topological structure antibacterial bone plate, comprise blade plate (1), it is characterized in that: described blade plate (1) surface coverage is kernel by Polyethylene Glycol-co-acroleic acid microgel core, outer controlled release shell is intelligent charge gradient radial distribution compound micelle (2) of pH responsive type polymethylacrylic acid microgel layer, and this compound micelle coating forms the compound micelle (2) on topological antibacterial structure surface at rustless steel, titanium or titanium alloy blade plate (1).
2. intelligent nano-hydrogel topological structure antibacterial bone plate according to claim 1, is characterized in that: described compound micelle (2) diameter about 100 ~ 400 μm, altitude range 0.6 ~ 8 μm.Polyethylene Glycol-co-acroleic acid microgel core potential range about negative 6 ~ 40mv, core internal diameter about 1 ~ 1000nm; The about negative 6 ~ 0mv of pH responsive type polymethylacrylic acid microgel layer potential range, the number of plies 5 ~ 30 layers.
3. intelligent nano-hydrogel topological structure antibacterial bone plate according to claim 1, is characterized in that: described blade plate (1) is rustless steel, titanium or titanium alloy.
4. intelligent nano-hydrogel topological structure antibacterial bone plate according to claim 3, is characterized in that: compound micelle (2) spacing range on described blade plate (1) upper topology antibacterial structure surface is 0.5 ~ 4 μm.
5. intelligent nano-hydrogel topological structure antibacterial bone plate according to claim 3, is characterized in that: the cationic antibacterial peptide of compound micelle (2) the load beta-alexin-1 ~ 3 on described blade plate (1) upper topology antibacterial structure surface, lactoferrin 1-11, LL-37, histatins analog, protegrins-1 and synthetic.
6. intelligent nano-hydrogel topological structure antibacterial bone plate according to claim 5, is characterized in that: the cationic antibacterial peptide per unit area concentration of compound micelle (2) the load beta-alexin-1 ~ 3 on described blade plate (1) upper topology antibacterial structure surface, lactoferrin 1-11, LL-37, histatins analog, protegrins-1 and synthetic is 0.025 ~ 1mg/mL.
7. a manufacture method for intelligent nano-hydrogel topological structure antibacterial bone plate as claimed in claim 1, is characterized in that: carry out as follows:
Step 1, titanium or (1) surface preparation of titanium alloy blade plate and the electric treatment of titanium surface band, titanium or titanium alloy blade plate (1) carry out hydrophilic treated each 10 ~ 20 minutes with distilled water, acetone, 75% ethanol, distilled water ultrasonic cleaning, panel successively, insert 0.2mg/mL, mean molecule quantity is 30 ~ 50k, many PLLs of pH9 2 ~ 3 hours, positively charged macromolecule layer is formed at titanium alloy substrate, carry out initial layer deposition, clean with deionized water after taking-up, at room temperature dried for standby;
Step 2, preparation PEGDA/AA emulsion, be averaged the polyethylene glycol acrylate 200 μ l of molecular weight 500 ~ 600 respectively, acrylic monomers leaves and takes 0 ~ 20 μ l after 50 DEG C of distillations, and light trigger 10 μ l, is jointly dissolved in dichloromethane 1ml and forms oil phase; 1 ~ 10% polyvinyl alcohol 1 ~ 3ml, as emulsifying agent, is dissolved in 10ml deionized water and forms aqueous phase; At high speed magneton with the stirring 10 ~ 20 minutes of 1000rpm, oil phase is dispersed in continuous print aqueous phase and forms emulsion, by adjusting the content of PVA, the ratio of oil phase/water phase, and the time that magneton stirs, controlling emulsion diameter is 1.0 ~ 1.5 μm;
Step 3, synthesis PEG-co-AA microgel core, the emulsion made is placed in lower 20 minutes of the uviol lamp of 100w.Ultraviolet excitation light trigger produces free radical, and under the principle of radical polymerization, the double bond of PEGDA and AA in oil droplet is opened, and causes cross-linking reaction between chain growth and chain; Therefore, PEGDA and AA in oil droplet defines microgel core, repeatedly carries out purification three times with 70% ethanol suspension-centrifugal method, removes ethanol by deionized water suspension-centrifugal method; By ratio 10:0.1 ~ 1 of control PEGDA and AA, control the charge density of microgel core, form electromotive force and be about the about negative 6 ~ 40mv of scope; The micelle core made is collected by 16000 ~ 50000r/min high speed centrifugation, and optionally can obtain required size 1 ~ 1000nm with the filter membrane of different pore size, the micelle core finally collected is dispersed in 10mlPBS phosphate buffer;
Step 4, PEG-co-AA microgel core finishing PMAA high charge density shell, first microgel core after purification be dispersed to the 0.2mg/mL of polyvinylpyrrolidone, pH 2, in 0.01M PBS solution, after 15 minutes, by microgel grain by filtering, centrifugal mode is cleaned, subsequently, the micelle of PVPON process is dispersed in the 0.2mg/mL of polymethylacrylic acid PMAA, in pH 2,0.01M PBS solution after 15 minutes, clean with said method, repeat above method 5 ~ 30 times, be formed with 5 ~ 30 layers of PVPON/PMAA gel layer of hydrogen bond crosslinks.
Step 5, cross-linked stable PMAA gel shell, micro-gel particles is dispersed in the 0.01MPBS of 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate 5mg/mL and hydroxy thiosuccinimide 5mg/mL, in the solution of PH5 1.5 hours; Afterwards, micelle is separated from solution, and is dipped into the ethylenediamine solution of 10mg/mL, within 14 hours, carry out between PMAA layer covalent cross-linking; Last PEG-co-AA/PMAA composite microgel grain to be soaked in the 0.01M PBS of pH7.5 2 hours, removes PVPON and other reagent unreacted, centrifugal-filter after composite microgel grain be suspended in PBS, in pH7.4,0.1M; The size of application dynamic light scattering and Zeta electric potential assay method composite microgel when different pH4 ~ 9 and ionic strength and charged situation, compound micelle (2) diameter about 100 ~ 400 μm, altitude range 0.6 ~ 8 μm.The about negative 6 ~ 0mv of PMAA microgel layer potential range, the number of plies about 5 ~ 30 layers;
Step 6, the structure on titanium topology antibacterial structure surface, microgel is in the deposition on titanium surface: compound micelle is suspended in the PBS of 0.01M, pH7.5, regulates micelle concentration to be 1.0 ~ 2.0 × 10
10to 10
11individual/ml.Many PLLs of positively charged positively charged macromolecule layer titanium sheet is dipped in above-mentioned microgel suspension, due to electrostatic adsorption, microgel by automatic absorbing to titanium plate surface, after 0.5 ~ 10 hour, titanium sheet is removed, alternately rinse with 0.01M PBS and deionized water, dry, the titanium sheet prepared is observed under scanning electron microscope, confirm the absorption of microgel, build and screen the surperficial titanium plate of antibacterial topological structure of gel diameter range 100 ~ 400 μm, altitude range 0.6 ~ 8 μm and gel interstice coverage 0.5 ~ 4 μm.
Step 7, the cationic antibacterial peptide of beta-alexin-1 ~ 3, lactoferrin 1-11LL-37, histatins analog, protegrins-1 and synthetic is loaded into the titanium surface of microgel modification.Under 36 ~ 37 DEG C of conditions, it is 10 ~ 20mL that the titanium surface that dry composite microgel is modified is soaked in concentration, concentration is 1mg/mL, pH value is the hBD-3 of 7.4, lactoferrin 1-11, LL-37, histatins analog, in the cationic antibacterial peptide PBS solution of protegrins-1 and synthetic, because charged character is contrary, hBD-3 is loaded in composite microgel, form year beta-alexin-1 ~ 3 with electric potential difference, lactoferrin 1-11, LL-37, histatins analog (histatin analogue DHVAR-5), the cationic antibacterial peptide topology antibacterial structure surface recombination micelle layer of protegrins-1 (Protegrins-1) and synthetic, dry under room temperature, obtain intelligent nano-hydrogel topological structure antibacterial bone plate.
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| CN111671970A (en) * | 2020-07-30 | 2020-09-18 | 齐鲁工业大学 | Polypeptide single-layer film with primary amino group exposure of 7%, and preparation method and application thereof |
| CN116083240A (en) * | 2023-04-07 | 2023-05-09 | 深圳市第二人民医院(深圳市转化医学研究院) | Engineered bacteria, preparation method and application thereof |
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| CN111671970A (en) * | 2020-07-30 | 2020-09-18 | 齐鲁工业大学 | Polypeptide single-layer film with primary amino group exposure of 7%, and preparation method and application thereof |
| CN111671970B (en) * | 2020-07-30 | 2021-12-14 | 齐鲁工业大学 | Polypeptide single-layer film with primary amino group exposure of 7%, and preparation method and application thereof |
| CN116083240A (en) * | 2023-04-07 | 2023-05-09 | 深圳市第二人民医院(深圳市转化医学研究院) | Engineered bacteria, preparation method and application thereof |
| CN116083240B (en) * | 2023-04-07 | 2023-08-29 | 深圳市第二人民医院(深圳市转化医学研究院) | Engineered bacteria, preparation method and application thereof |
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