CN105288748A - Anti-infection calcium phosphate composite bone cement material and preparation method thereof - Google Patents
Anti-infection calcium phosphate composite bone cement material and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an anti-infection calcium phosphate composite bone cement material and a preparation method thereof. The preparation method comprises the following steps: enabling gamma-polyglutamic acid modified by phosphorylcholine to adsorb nano-silver, and carrying out graft copolymerization reaction between water-soluble polysaccharide and gamma-polyglutamic acid to prepare a nano-silver anti-infection agent with an anti-infection effect; and on the basis that the basic characteristics of calcium phosphate bone cement are not affected, compounding and blending the anti-infection agent and other assistants with medical calcium phosphate bone cement, and then preparing the calcium phosphate composite bone cement material with an anti-infection function by adopting a molding method. The method disclosed by the invention is simple in process, and the compressive strength of the prepared calcium phosphate composite bone cement material reaches 70MPa; the calcium phosphate composite bone cement material does not have obvious cytotoxicity and blood coagulation to osteoblasts and bone marrow stromal cells and does not have obvious sensitization, stimulation and genetic toxicity; and the calcium phosphate composite bone cement material has a wide antibacterial spectrum and has certain antibacterial effects on Gram-positive cocci and Gram-negative bacilli, and the antibacterial rate reaches 98%.
Description
Technical field
The invention belongs to technical field of biological materials, be specifically related to a kind of infection calcium phosphate composite bone cement material and preparation method thereof.
Background technology
Biomaterial and products thereof in use, run into some extent with body tissue can not completely compatible, infect, canceration, biological organization material the series of problems such as calcification.That wherein applies along with biomaterial with the infection (BRI) that biomaterial is relevant increases, and sickness rate obviously increases, and preventing and treating with biomaterial associated infection is clinically face urgent problem.A lot of research shows that implantable bioartificial material adds bacterial invasion host approach, reduces the defence capability of body, reduces the effect stoping bacterial adhesion and biofilm formation.Biomembrane is once be formed, and for free bacteria provides new attachment sites, antibacterial constantly dissociates or discharges from biomembrane simultaneously, becomes chronic infection source.Therefore, the method for research control biomaterial bacterial adhesion becomes the study hotspot in this field.Calcium phosphate bone cement (CPC) is a kind of ceramic material with good biological activity and biocompatibility, the fields such as orthopaedics, surgery, the department of stomatology are widely used in, because having good histocompatibility, biological degradability and bone guided active, having certain comprcssive strength, is good bone alternate material clinically.But the problems such as the organism infection that calcium phosphate bone cement implant causes, greatly limit its range of application and value.
At present, external employing solves at biomedical devices top finishing anti-infective or antibiotic method the infection that biomaterial for medical purpose implants relationship type, serves certain effect.But find that the anti-infection bio material medical apparatus and instruments prepared by covering with paint anti-infective or antibiotic has following shortcoming in actual use: (1) application type coating, under the immersion of body fluid, may depart from from biomaterial material surface thus lose anti-infectious function.And stratum disjunction may affect the health of human body.(2) the coating useful effect phase is short, cannot ensure anti-infection property during life-time service.(3) the anti-infective medical apparatus and instruments preparing coating has to pass through coating operation when making, and production technology needs increase equipment, adds extra cost of manufacture.(4) when the biomaterial medical apparatus and instruments that preparation structure is more complicated, cover with paint, lacquer, colour wash, etc. infection layer and often cannot realize in technique, that is Coating technology is by goods structural limitations.
Summary of the invention
The object of the invention is the shortcomings such as, vulnerability large, reliability low, narrow application range short for prior art floating coat type medical anti-infectious biomaterial effect duration, provide a kind of infection dependable performance, effective acting time long, has a broad antifungal spectrum and the strong infection calcium phosphate composite bone cement material of adaptability for materials and preparation method thereof.
The means that anti-infective is added in the inventive method employing in biomaterial have prepared overall medical anti-infectious biomaterial---the infection calcium phosphate composite bone cement material with infection and antibacterium adhesive function.
First object of the present invention is to provide the preparation method of infection calcium phosphate composite bone cement material, it is characterized in that, comprises the following steps:
Raw material: by gross weight 100 parts, comprises gamma-polyglutamic acid-nanometer silver anti-infective 0.01 ~ 10 weight portion, binding agent 0 ~ 20 weight portion, dispersant 0 ~ 10 weight portion, the medical calcium phosphate bone cement pressed powder of stabilizing agent 0 ~ 5 weight portion and surplus;
Each component is taken by the weight portion of each component of above-mentioned raw materials, gamma-polyglutamic acid-nanometer silver anti-infective, binding agent, dispersant and stabilizing agent is mixed in medical calcium phosphate bone cement pressed powder, then abundant mix homogeneously, using water or concentration 0.01 ~ 1.0g/L gamma-polyglutamic acid-aqueous solution as distiller liquor, be in harmonious proportion by the liquid-solid ratio of L/P=0.33 ~ 0.4ml/g, after solidification, obtain infection calcium phosphate composite bone cement material;
Described gamma-polyglutamic acid-nanometer silver anti-infective is prepared by the following method:
Utilize phosphocholine to modify gamma-polyglutamic acid-, then by the gamma-polyglutamic acid-of phosphocholine modified absorption nanometer silver, then add water soluble polysaccharide wherein and gamma-polyglutamic acid-carries out graft copolymerization, prepare gamma-polyglutamic acid-nanometer silver anti-infective.
Preferably, described gamma-polyglutamic acid-nanometer silver anti-infective is prepared by the following method:
(1) be that the gamma-polyglutamic acid-of 1:1 ~ 10 and phosphocholine are placed in dehydrated alcohol by mass ratio, and stirring reaction 12 ~ 18h under dehydrated alcohol environment all the time, centrifugal, abandon supernatant, lyophilization obtains gamma-polyglutamic acid--phosphocholine; Gained gamma-polyglutamic acid--phosphocholine is dissolved in anhydrous dimethyl sulphoxide, is prepared into the gamma-polyglutamic acid--phosphocholine solution of 5 ~ 50g/L;
(2) in gamma-polyglutamic acid--phosphocholine solution: nanometer silver solution volume ratio is that 0.1 ~ 5.0mmol/L nanometer silver solution adds in gamma-polyglutamic acid--phosphocholine solution by the ratio of 2 ~ 0.5:1, in lucifuge situation, carry out stirring 5 ~ 12h, obtain gamma-polyglutamic acid-nanometer silver solution thus;
(3) in gamma-polyglutamic acid-nanometer silver solution, catalytic materials is added, its final concentration is made to be 0.002 ~ 0.01mmol/L, then the water soluble polysaccharide solution that concentration is 0.01 ~ 0.5g/L, pH value is 5 ~ 7 is added under agitation gradually, the volume ratio of water soluble polysaccharide solution and gamma-polyglutamic acid-nanometer silver solution is 1 ~ 5:1,20 ~ 60 DEG C are stirred 3 ~ 24h and carry out graft copolymerization formation precipitated product, stopped reaction, add acetone and stir precipitated product, get precipitation, by distilled water wash precipitation, namely lyophilization obtains gamma-polyglutamic acid-nanometer silver anti-infective.
Described catalytic materials is 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDCHCl), dicyclohexylcarbodiimide (DCC), I-hydroxybenzotriazole (HOBT), 1-hydroxyl-7-azo BTA (HOAT), 3-hydroxyl-1, the compositions of one or two or more kinds in 2,3-phentriazine-4 (3H)-one (HOOBt), N-hydroxy-succinamide (NHS) and DMAP (DMAP).
Described water soluble polysaccharide comprises plant water-soluble polysaccharide, animal water soluble polysaccharide, microorganism water soluble polysaccharide or marine polysaccharide: plant water-soluble polysaccharide includes tea polysaccharide, lycium barbarum polysaccharide, konjacmannan, Ginkgo biloba polysaccharide, Semen Ginkgo extracellular polysaccharide, lentinan, tremella polysaccharide, ganoderan, Auricularia polycose, pachyman, Taraxacum Polysaccharides, soluble starch, water-soluble fibre; Animal water soluble polysaccharide comprises mucopolysaccharide, heparin, chondroitin sulfate, hyaluronic acid etc.; Microorganism water soluble polysaccharide comprises lentinan, pachyman, tremella polysaccharide, krestin etc.; Marine polysaccharide comprises soluble chitin, spirulina polysaccharide; The aqueous solution that described water soluble polysaccharide solution is is solute with one or two or more kinds above-mentioned water soluble polysaccharide.
Described binding agent is gamma-polyglutamic acid-/chitosan.Wherein gamma-polyglutamic acid-/chitosan adopts document (to dredge elegant woods, Shi Qingshan, Lin Xiaoping, Ou Yangyousheng, Chen Yiben. the preparation of gamma-polyglutamic acid-/chitosan multi-porous compound support frame material, the research of sign and performance, research and development of natural products, 2013, 25:514-518) described preparation method obtains, its concrete grammar example is as follows: take 0.5g gamma-polyglutamic acid-hydrogen salt (γ-PGA) and 0.05g4-dimethylamino naphthyridine ultrasonic dissolution in 18 milliliters of anhydrous dimethyl sulphoxides, Carboxy Chitosan solution is slowly added drop-wise in γ-PGA solution, and with magnetic agitation, dropwise and add a certain amount of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, experiment is stopped after stirred at ambient temperature 3h.The long-pending acetone of triploid will be added in mixed solution, to form precipitation, vacuum filtration collecting precipitation, precipitate is carried out lyophilization, remove unnecessary acetone, then precipitate PBS solvent (pH value 7.2) is dissolved, use ultrafiltration dialysis method to remove unnecessary chitosan (bag filter molecular cut off 100,000) and 1,6-unnecessary ethylenediamine (bag filter molecular cut off 3500).Gradient freezing: with 4 DEG C ,-20 DEG C each 2h ,-40 DEG C of 4h ,-70 DEG C of 24h, carry out vacuum lyophilization 48h afterwards, vacuum reaches 0.01Pa, obtains gamma-polyglutamic acid-/chitosan, is ground into powder and obtains gamma-polyglutamic acid-/chitosan freeze-dried powder and use.
Described dispersant is the compositions of one or two or more kinds in gamma-polyglutamic acid-, Polyethylene Glycol, stearic amide class dispersant or stearic acid dispersant or polyethylene.
Described stabilizing agent is antioxidant 168, polyvinyl alcohol or rare-earth stabilizer (XT-1, XT-2, XT-3).
The consumption of described acetone is 2 ~ 4 times of reactant liquor volume.
Second object of the present invention is to provide a kind of infection calcium phosphate composite bone cement material, it is characterized in that, its raw material is: by gross weight 100 parts, comprise gamma-polyglutamic acid-nanometer silver anti-infective 0.01 ~ 10 weight portion, binding agent 0 ~ 20 weight portion, dispersant 0 ~ 10 weight portion, the medical calcium phosphate bone cement pressed powder of stabilizing agent 0 ~ 5 weight portion and surplus;
Described gamma-polyglutamic acid-nanometer silver anti-infective is prepared by the following method:
Utilize phosphocholine to modify gamma-polyglutamic acid-, then by the gamma-polyglutamic acid-of phosphocholine modified absorption nanometer silver, then add water soluble polysaccharide wherein and gamma-polyglutamic acid-carries out graft copolymerization, prepare gamma-polyglutamic acid-nanometer silver anti-infective.
The present invention have selected and do not destroy calcium phosphate bone cement primary characteristic, suitable macromolecule anti-infective and other auxiliary agent and composite, the blended rear self-curing of medical calcium phosphate bone cement material are prepared into the calcium phosphate bone cement material with infection function, under the prerequisite keeping current medical anti-infectious calcium phosphate bone cement fundamental characteristics, solve calcium phosphate bone cement and cause the problems such as matrix infection.Therefore the medical anti-infectious calcium phosphate composite bone cement material in the present invention has infection dependable performance, the advantages such as effective acting time is long, has a broad antifungal spectrum, and adaptability for materials is strong.
Detailed description of the invention
Following examples further illustrate of the present invention, instead of limitation of the present invention.
The binding agent used in following examples is gamma-polyglutamic acid-/chitosan.Wherein gamma-polyglutamic acid-/chitosan adopts document (to dredge elegant woods, Shi Qingshan, Lin Xiaoping, Ou Yangyousheng, Chen Yiben. the preparation of gamma-polyglutamic acid-/chitosan multi-porous compound support frame material, the research of sign and performance, research and development of natural products, 2013, 25:514-518) described preparation method obtains, its concrete grammar example is as follows: take 0.5g gamma-polyglutamic acid-hydrogen salt (γ-PGA) and 0.05g4-dimethylamino naphthyridine ultrasonic dissolution in 18 milliliters of anhydrous dimethyl sulphoxides, Carboxy Chitosan solution is slowly added drop-wise in γ-PGA solution, and with magnetic agitation, dropwise and add a certain amount of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, experiment is stopped after stirred at ambient temperature 3h.The long-pending acetone of triploid will be added in mixed solution, to form precipitation, vacuum filtration collecting precipitation, precipitate is carried out lyophilization, remove unnecessary acetone, then precipitate PBS solvent (pH value 7.2) is dissolved, use ultrafiltration dialysis method to remove unnecessary chitosan (bag filter molecular cut off 100,000) and 1,6-unnecessary ethylenediamine (bag filter molecular cut off 3500).Gradient freezing: with 4 DEG C ,-20 DEG C each 2h ,-40 DEG C of 4h ,-70 DEG C of 24h, carry out vacuum lyophilization 48h afterwards, vacuum reaches 0.01Pa, obtains gamma-polyglutamic acid-/chitosan, is ground into powder and obtains gamma-polyglutamic acid-/chitosan freeze-dried powder and use.
Embodiment 1:
A kind of infection calcium phosphate composite bone cement material, prepared by following steps:
(1) gamma-polyglutamic acid-and phosphocholine is taken, the mass ratio of gamma-polyglutamic acid-and phosphocholine is 1:1, gamma-polyglutamic acid-and phosphocholine are placed in enough dehydrated alcohol, do not stop to stir 12h and (beaker adds preservative film sealing, prevent ethanol from volatilizing, thus reaction is carried out all the time under dehydrated alcohol environment), then by mixed solution with 4 × 10
3the centrifugal 2min of rpm, abandons supernatant, finds time, volatilizees, lyophilization obtains gamma-polyglutamic acid--phosphocholine white powder.
(2) by step (1) gained gamma-polyglutamic acid--phosphocholine white powder ultrasonic dissolution in anhydrous dimethyl sulphoxide, be prepared into the gamma-polyglutamic acid--phosphocholine solution of 5.0g/L, seal for subsequent use.Configuration 0.1mmol/L nanometer silver solution, in gamma-polyglutamic acid--phosphocholine solution: nanometer silver solution volume ratio is that nanometer silver solution adds in gamma-polyglutamic acid--phosphocholine solution by the ratio of 2:1, carries out stirring 5h in lucifuge situation.Obtain gamma-polyglutamic acid-nanometer silver solution thus.
(3) in step (2) gained gamma-polyglutamic acid-nanometer silver solution, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDCHCl) and DMAP (DMAP) is added, its final concentration is made to be respectively 0.001mmol/L and 0.001mmol/L, then the soluble starch solution (pH value is 5) that concentration is 0.01g/L is dropwise added under agitation, the volume ratio of soluble starch solution and gamma-polyglutamic acid-nanometer silver solution is 1:1, 40 DEG C are stirred 3h and carry out graft copolymerization formation precipitated product, stopped reaction, the acetone adding 2 times of reactant liquor volumes stirs precipitated product, get precipitation, 3 times are precipitated with distilled water wash, namely lyophilization obtains gamma-polyglutamic acid-nanometer silver anti-infective.
(4) in the clean environment meeting GMP requirement, count by weight, 10 parts of gamma-polyglutamic acid-nanometer silver anti-infectives are mixed in 90 parts of medical calcium phosphate bone cement pressed powders, and fully mix in clean high-speed stirred unit, be that 0.01g/L gamma-polyglutamic acid-aqueous solution is as distiller liquor with concentration, be in harmonious proportion by the liquid-solid ratio of L/P=0.33ml/g, after solidification, obtain infection calcium phosphate composite bone cement material.
Mechanical strength: carry out intensity test according to GB/T4740-1999 " ceramic material compressive strength test method " described experimental technique, experimental result shows, the comprcssive strength of the infection calcium phosphate composite bone cement material that the present embodiment obtains is 72+10MPa.
Biological safety: carry out biological assessment according to GB/T16886 " BiologicalEvaluationofMedicalDevice " described experimental technique.Experimental result shows, the obtained infection calcium phosphate composite bone cement material of the present embodiment does not have obvious cytotoxicity and hemagglutinin to osteoblast and bone marrow stroma stem cell, does not have obvious sensitization, stimulation and genetoxic.
Anti-microbial property: adopt following experimental strain: 1. S. epdermidis strains (StaphylococcusepidermidisATCC12228), 2. staphylococcus aureus (StaphylococcusaureuATCC25923), 3. Pseudomonas aeruginosa (PseudomonasaeruginosaATCC27853) and 4. escherichia coli (EscherichiacoliATCC25922); Bacteriostatic experiment is according to standard regulations such as " JISZ2801-2000 " antibacterial fabricated product-antibiotic property test method and antibacterial effect ", GB/T21510-2008 " nano inorganic material anti-microbial property detection method " ".Result shows, the infection calcium phosphate composite bone cement material that the present embodiment obtains to 1., 2., 3., 4. the antibiotic rate of number bacterial strain be respectively 98%, 85%, 90%, 70%, the antibiotic rate of matched group (traditional calcium phosphate bone cement) is 0%.
Embodiment 2:
A kind of infection calcium phosphate composite bone cement material, prepared by following steps:
(1) gamma-polyglutamic acid-and phosphocholine is taken, the mass ratio of gamma-polyglutamic acid-and phosphocholine is 1:2, gamma-polyglutamic acid-and phosphocholine are placed in dehydrated alcohol, do not stop to stir 18h and (beaker adds preservative film sealing, prevent ethanol from volatilizing, thus reaction is carried out all the time under dehydrated alcohol environment), then by mixed solution with 4 × 10
3the centrifugal 10min of rpm, abandons supernatant, finds time, volatilizees, lyophilization obtains gamma-polyglutamic acid--phosphocholine white powder.
(2) by step (1) gained gamma-polyglutamic acid--phosphocholine white powder ultrasonic dissolution in anhydrous dimethyl sulphoxide, be prepared into the gamma-polyglutamic acid--phosphocholine solution of 10g/L, seal for subsequent use.Configuration 1.0mmol/L nanometer silver solution, in gamma-polyglutamic acid--phosphocholine solution: nanometer silver solution volume ratio is that nanometer silver solution adds in gamma-polyglutamic acid--phosphocholine solution by the ratio of 1:1, carries out stirring 10h in lucifuge situation.Obtain gamma-polyglutamic acid-nanometer silver solution thus.
(3) in step (2) gained gamma-polyglutamic acid-nanometer silver solution, dicyclohexylcarbodiimide (DCC) and I-hydroxybenzotriazole (HOBT) is added, its final concentration is made to be respectively 0.005mmol/L and 0.001mmol/L, then the Ginkgo biloba polysaccharide solution (pH value is 6.0) that concentration is 0.10g/L is dropwise added under agitation, the volume ratio of Ginkgo biloba polysaccharide solution and gamma-polyglutamic acid-nanometer silver solution is 2:1, 20 DEG C are stirred 12h and carry out graft copolymerization formation precipitated product, stopped reaction, the acetone adding 3 times of reactant liquor volumes stirs precipitated product, get precipitation, 4 times are precipitated with distilled water wash, namely lyophilization obtains gamma-polyglutamic acid-nanometer silver anti-infective.
(4) in the clean environment meeting GMP requirement, count by weight, 0.01 part of gamma-polyglutamic acid-nanometer silver anti-infective is mixed in 87.99 parts of medical calcium phosphate bone cement pressed powders, 2 parts of binding agents (gamma-polyglutamic acid-/chitosan freeze-dried powder), 5 parts of dispersants (Polyethylene Glycol) and 5 parts of stabilizing agents (rare-earth stabilizer XT-1), and fully mix in clean high-speed stirred unit, using concentration 0.05g/L gamma-polyglutamic acid-aqueous solution as distiller liquor, be in harmonious proportion by the liquid-solid ratio of L/P=0.35ml/g, infection calcium phosphate composite bone cement material is obtained after solidification.
Mechanical strength: carry out intensity test according to GB/T4740-1999 " ceramic material compressive strength test method " described experimental technique, experimental result shows, the comprcssive strength of the infection calcium phosphate composite bone cement material that the present embodiment obtains is 55+7MPa.
Biological safety: carry out biological assessment according to GB/T16886 " BiologicalEvaluationofMedicalDevice " described experimental technique.Experimental result shows, the obtained infection calcium phosphate composite bone cement material of the present embodiment does not have obvious cytotoxicity and hemagglutinin to osteoblast and bone marrow stroma stem cell, does not have obvious sensitization, stimulation and genetoxic.
Anti-microbial property: adopt following experimental strain: 1. S. epdermidis strains (StaphylococcusepidermidisATCC12228), 2. staphylococcus aureus (StaphylococcusaureuATCC25923), 3. Pseudomonas aeruginosa (PseudomonasaeruginosaATCC27853) and 4. escherichia coli (EscherichiacoliATCC25922); Bacteriostatic experiment is according to standard regulations such as " JISZ2801-2000 " antibacterial fabricated product-antibiotic property test method and antibacterial effect ", GB/T21510-2008 " nano inorganic material anti-microbial property detection method " ".Result show, the infection calcium phosphate composite bone cement material that the present embodiment obtains to 1., 2., 3., 4. the antibiotic rate of number bacterial strain be respectively 53%, 47%, 56%, 39%, the antibiotic rate of matched group (traditional calcium phosphate bone cement) is 0%.
Embodiment 3:
A kind of infection calcium phosphate bone composite cement material, prepared by following steps:
(1) gamma-polyglutamic acid-and phosphocholine is taken, the mass ratio of gamma-polyglutamic acid-and phosphocholine is 1:4, gamma-polyglutamic acid-and phosphocholine are placed in dehydrated alcohol, do not stop to stir 12h and (beaker adds preservative film sealing, prevent ethanol from volatilizing, thus reaction is carried out all the time under dehydrated alcohol environment), then by mixed solution with 4 × 10
3the centrifugal 15min of rpm, abandons supernatant, finds time, volatilizees, lyophilization obtains gamma-polyglutamic acid--phosphocholine white powder.
(2) by step (1) gained gamma-polyglutamic acid--phosphocholine white powder ultrasonic dissolution in anhydrous dimethyl sulphoxide, be prepared into the gamma-polyglutamic acid--phosphocholine solution of 50g/L, seal for subsequent use.Configuration 5.0mmol/L nanometer silver solution, in gamma-polyglutamic acid--phosphocholine solution: nanometer silver solution volume ratio is that nanometer silver solution adds in gamma-polyglutamic acid--phosphocholine solution by the ratio of 1:2, carries out stirring 12h in lucifuge situation.Obtain gamma-polyglutamic acid-nanometer silver solution thus.
(3) in step (2) gained gamma-polyglutamic acid-nanometer silver solution, adding N-hydroxy-succinamide (NHS) makes its final concentration be 0.01mmol/L, then the chondroitin sulfate solution (pH value is 7.0) that concentration is 0.5g/L is dropwise added under agitation, the volume ratio of chondroitin sulfate solution and gamma-polyglutamic acid-nanometer silver solution is 5:1, 60 DEG C are stirred 24h and carry out graft copolymerization formation precipitated product, stopped reaction, the acetone adding 4 times of reactant liquor volumes stirs precipitated product, get precipitation, 5 times are precipitated with distilled water wash, namely lyophilization obtains gamma-polyglutamic acid-nanometer silver anti-infective.
(4) in the clean environment meeting GMP requirement, count by weight, 0.5 part of gamma-polyglutamic acid-nanometer silver anti-infective is mixed in 71.5 parts of medical calcium phosphate bone cement pressed powders, 20 parts of binding agents (gamma-polyglutamic acid-/chitosan freeze-dried powder), 3 parts of dispersants (stearic amide) and 5 parts of stabilizing agents (polyvinyl alcohol), and fully mix in clean high-speed stirred unit, using concentration 1.0g/L gamma-polyglutamic acid-aqueous solution as distiller liquor, be in harmonious proportion by the liquid-solid ratio of L/P=0.38ml/g, infection calcium phosphate composite bone cement material is obtained after solidification.
Mechanical strength: carry out intensity test according to GB/T4740-1999 " ceramic material compressive strength test method " described experimental technique, experimental result shows, the comprcssive strength of the infection calcium phosphate composite bone cement material that the present embodiment obtains is 61 ± 5MPa.
Biological safety: carry out biological assessment according to GB/T16886 " BiologicalEvaluationofMedicalDevice " described experimental technique, experimental result shows, the obtained infection calcium phosphate composite bone cement of the present embodiment does not have obvious cytotoxicity and hemagglutinin to material osteoblast and bone marrow stroma stem cell, does not have obvious sensitization, stimulation and genetoxic.
Anti-microbial property: adopt following experimental strain: 1. S. epdermidis strains (StaphylococcusepidermidisATCC12228), 2. staphylococcus aureus (StaphylococcusaureuATCC25923), 3. Pseudomonas aeruginosa (PseudomonasaeruginosaATCC27853) and 4. escherichia coli (EscherichiacoliATCC25922); Bacteriostatic experiment is according to standard regulations such as " JISZ2801-2000 " antibacterial fabricated product-antibiotic property test method and antibacterial effect ", GB/T21510-2008 " nano inorganic material anti-microbial property detection method " ".Result shows, the infection calcium phosphate composite bone cement material that the present embodiment obtains to 1., 2., 3., 4. the antibiotic rate of number bacterial strain be respectively 76%, 71%, 84%, 47%, the antibiotic rate of matched group (traditional calcium phosphate bone cement) is 0%.
Embodiment 4:
A kind of infection calcium phosphate composite bone cement material, prepared by following steps:
(1) gamma-polyglutamic acid-and phosphocholine is taken, the mass ratio of gamma-polyglutamic acid-and phosphocholine is 1:3, gamma-polyglutamic acid-and phosphocholine are placed in dehydrated alcohol, do not stop to stir 12h and (beaker adds preservative film sealing, prevent ethanol from volatilizing, thus reaction is carried out all the time under dehydrated alcohol environment), then by mixed solution with 4 × 10
3the centrifugal 2min of rpm, abandons supernatant, finds time, volatilizees, lyophilization obtains gamma-polyglutamic acid--phosphocholine white powder.
(2) by step (1) gained gamma-polyglutamic acid--phosphocholine white powder ultrasonic dissolution in anhydrous dimethyl sulphoxide, be prepared into the gamma-polyglutamic acid--phosphocholine solution of 15g/L, seal for subsequent use.Configuration 2.5mmol/L nanometer silver solution, in gamma-polyglutamic acid--phosphocholine solution: nanometer silver solution volume ratio is that nanometer silver solution adds in gamma-polyglutamic acid--phosphocholine solution by the ratio of 1:1.5, carries out stirring 10h in lucifuge situation.Obtain gamma-polyglutamic acid-nanometer silver solution thus.
(3) in step (2) gained gamma-polyglutamic acid-nanometer silver solution, 1-hydroxyl-7-azo BTA (HOAT) and 3-hydroxyl-1 is added, 2, 3-phentriazine-4 (3H)-one (HOOBt) makes its final concentration be respectively 0.075mmol/L and 0.001mmol/L, then the spirulina polysaccharide solution (pH value is 6.0) that concentration is 0.30g/L is dropwise added under agitation, the volume ratio of spirulina polysaccharide solution and gamma-polyglutamic acid-nanometer silver solution is 4:1, 60 DEG C are stirred 18h and carry out graft copolymerization formation precipitated product, stopped reaction, the acetone adding 2 times of reactant liquor volumes stirs precipitated product, get precipitation, stabilizing agent is added after precipitating 3 times with distilled water wash, stir, namely lyophilization obtains gamma-polyglutamic acid-nanometer silver anti-infective.
(4) in the clean environment meeting GMP requirement, count by weight, 2 parts of gamma-polyglutamic acid-nanometer silver anti-infectives, 5 parts of binding agents (gamma-polyglutamic acid-/chitosan freeze-dried powder), 10 parts of dispersants (polyethylene) and 5 parts of stabilizing agents (rare-earth stabilizer XT-2) are mixed in 78 parts of medical calcium phosphate bone cement pressed powders, and fully mix in clean high-speed stirred unit, using water as distiller liquor, be in harmonious proportion by the liquid-solid ratio of L/P=0.4ml/g, after solidification, obtain infection calcium phosphate composite bone cement material.
Mechanical strength: carry out intensity test according to GB/T4740-1999 " ceramic material compressive strength test method " described experimental technique, experimental result shows, the comprcssive strength of the infection calcium phosphate composite bone cement material that the present embodiment obtains is 55 ± 8MPa.
Biological safety: carry out biological assessment according to GB/T16886 " BiologicalEvaluationofMedicalDevice " described experimental technique, experimental result shows, the obtained infection calcium phosphate composite bone cement material of the present embodiment does not have obvious cytotoxicity and hemagglutinin to osteoblast and bone marrow stroma stem cell, does not have obvious sensitization, stimulation and genetoxic.
Anti-microbial property: adopt following experimental strain: 1. S. epdermidis strains (StaphylococcusepidermidisATCC12228), 2. staphylococcus aureus (StaphylococcusaureuATCC25923), 3. Pseudomonas aeruginosa (PseudomonasaeruginosaATCC27853) and 4. escherichia coli (EscherichiacoliATCC25922); Bacteriostatic experiment is according to standard regulations such as " JISZ2801-2000 " antibacterial fabricated product-antibiotic property test method and antibacterial effect ", GB/T21510-2008 " nano inorganic material anti-microbial property detection method " ".Result shows, the infection calcium phosphate composite bone cement material that the present embodiment obtains to 1., 2., 3., 4. the antibiotic rate of number bacterial strain be respectively 87%, 73%, 56%, 88%, the antibiotic rate of matched group (traditional calcium phosphate bone cement) is 0%.
Claims (10)
1. a preparation method for infection calcium phosphate composite bone cement material, is characterized in that, comprises the following steps:
Raw material: by gross weight 100 parts, comprises gamma-polyglutamic acid-nanometer silver anti-infective 0.01 ~ 10 weight portion, binding agent 0 ~ 20 weight portion, dispersant 0 ~ 10 weight portion, the medical calcium phosphate bone cement pressed powder of stabilizing agent 0 ~ 5 weight portion and surplus;
Each component is taken by the weight portion of each component of above-mentioned raw materials, gamma-polyglutamic acid-nanometer silver anti-infective, binding agent, dispersant and stabilizing agent is mixed in medical calcium phosphate bone cement pressed powder, then abundant mix homogeneously, using water or concentration 0.01 ~ 1.0g/L gamma-polyglutamic acid-aqueous solution as distiller liquor, be in harmonious proportion by the liquid-solid ratio of L/P=0.33 ~ 0.4ml/g, after solidification, obtain infection calcium phosphate composite bone cement material;
Described gamma-polyglutamic acid-nanometer silver anti-infective is prepared by the following method:
Utilize phosphocholine to modify gamma-polyglutamic acid-, then by the gamma-polyglutamic acid-of phosphocholine modified absorption nanometer silver, then add water soluble polysaccharide wherein and gamma-polyglutamic acid-carries out graft copolymerization, prepare gamma-polyglutamic acid-nanometer silver anti-infective.
2. preparation method according to claim 1, is characterized in that, described gamma-polyglutamic acid-nanometer silver anti-infective is prepared by the following method:
(1) be that the gamma-polyglutamic acid-of 1:1 ~ 10 and phosphocholine are placed in dehydrated alcohol by mass ratio, and stirring reaction 12 ~ 18h under dehydrated alcohol environment all the time, centrifugal, abandon supernatant, lyophilization obtains gamma-polyglutamic acid--phosphocholine; Gained gamma-polyglutamic acid--phosphocholine is dissolved in anhydrous dimethyl sulphoxide, is prepared into the gamma-polyglutamic acid--phosphocholine solution of 5 ~ 50g/L;
(2) in gamma-polyglutamic acid--phosphocholine solution: nanometer silver solution volume ratio is that 0.1 ~ 5.0mmol/L nanometer silver solution adds in gamma-polyglutamic acid--phosphocholine solution by the ratio of 2 ~ 0.5:1, in lucifuge situation, carry out stirring 5 ~ 12h, obtain gamma-polyglutamic acid-nanometer silver solution thus;
(3) in gamma-polyglutamic acid-nanometer silver solution, catalytic materials is added, its final concentration is made to be 0.002 ~ 0.01mmol/L, then adding concentration under agitation is gradually 0.01 ~ 0.5g/L, pH value is the water soluble polysaccharide solution of 5 ~ 7, the volume ratio of water soluble polysaccharide solution and gamma-polyglutamic acid-nanometer silver solution is 1 ~ 5:1, 20 ~ 60 DEG C are stirred 3 ~ 24h and carry out graft copolymerization formation precipitated product, stopped reaction, add acetone and stir precipitated product, get precipitation, precipitate with distilled water wash, then namely lyophilization obtains gamma-polyglutamic acid-nanometer silver anti-infective.
3. preparation method according to claim 2, it is characterized in that, described catalytic materials is 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDCHCl), dicyclohexylcarbodiimide (DCC), I-hydroxybenzotriazole (HOBT), 1-hydroxyl-7-azo BTA (HOAT), 3-hydroxyl-1, the compositions of one or two or more kinds in 2,3-phentriazine-4 (3H)-one (HOOBt), N-hydroxy-succinamide (NHS) and DMAP (DMAP).
4. preparation method according to claim 2, is characterized in that, described water soluble polysaccharide is plant water-soluble polysaccharide, animal water soluble polysaccharide, microorganism water soluble polysaccharide or marine polysaccharide.
5. preparation method according to claim 4, it is characterized in that, described plant water-soluble polysaccharide comprises tea polysaccharide, lycium barbarum polysaccharide, konjacmannan, Ginkgo biloba polysaccharide, Semen Ginkgo extracellular polysaccharide, lentinan, tremella polysaccharide, ganoderan, Auricularia polycose, pachyman, Taraxacum Polysaccharides, soluble starch and water-soluble fibre; Described animal water soluble polysaccharide comprises mucopolysaccharide, heparin, chondroitin sulfate and hyaluronic acid; Described microorganism water soluble polysaccharide comprises lentinan, pachyman, tremella polysaccharide and krestin; Described marine polysaccharide comprises soluble chitin, spirulina polysaccharide; The aqueous solution that described water soluble polysaccharide solution is is solute with one or two or more kinds above-mentioned water soluble polysaccharide.
6. preparation method according to claim 2, is characterized in that, the consumption of described acetone is 2 ~ 4 times of reactant liquor volume.
7. preparation method according to claim 1 and 2, is characterized in that, described binding agent is gamma-polyglutamic acid-/chitosan.
8. preparation method according to claim 1 and 2, is characterized in that, described dispersant is the compositions of one or two or more kinds in gamma-polyglutamic acid-, Polyethylene Glycol, stearic amide class dispersant or stearic acid dispersant or polyethylene.
9. preparation method according to claim 1 and 2, is characterized in that, described stabilizing agent is antioxidant 168, polyvinyl alcohol or rare-earth stabilizer.
10. an infection calcium phosphate composite bone cement material, it is characterized in that, its raw material is: by gross weight 100 parts, comprise gamma-polyglutamic acid-nanometer silver anti-infective 0.01 ~ 10 weight portion, binding agent 0 ~ 20 weight portion, dispersant 0 ~ 10 weight portion, the medical calcium phosphate bone cement pressed powder of stabilizing agent 0 ~ 5 weight portion and surplus;
Described gamma-polyglutamic acid-nanometer silver anti-infective is prepared by the following method:
Utilize phosphocholine to modify gamma-polyglutamic acid-, then by the gamma-polyglutamic acid-of phosphocholine modified absorption nanometer silver, then add water soluble polysaccharide wherein and gamma-polyglutamic acid-carries out graft copolymerization, prepare gamma-polyglutamic acid-nanometer silver anti-infective.
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