CN102727424B - Sodium hyaluronate gel injection for bone joint cavity and preparation method thereof - Google Patents
Sodium hyaluronate gel injection for bone joint cavity and preparation method thereof Download PDFInfo
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Abstract
The invention discloses sodium hyaluronate gel injection for a bone joint cavity and preparation methods of the sodium hyaluronate gel injection. One preparation method for the sodium hyaluronate gel injection comprises the following steps of: preparing crosslinked sodium hyaluronate gel and sodium hyaluronate gel, and uniformly mixing and sterilizing the crosslinked sodium hyaluronate gel and the sodium hyaluronate gel; and the other preparation method for the sodium hyaluronate gel injection comprises the following steps of: preparing dry crosslinked sodium hyaluronate gel powder, mixing the dry crosslinked sodium hyaluronate gel powder and dry sodium hyaluronate gel powder uniformly to obtain mixed powder, swelling, purifying and sterilizing. The cross linker residue in the sodium hyaluronate gel injection for the bone joint cavity is lower than 2ppm; after the injection is injected to the bone joint cavity, toxic and side effects produced by toxicity of a cross linker are avoided, the degradation speed is low, and the retention time of the injection in the joint cavity can reach 6 months under the normal condition.
Description
Technical field
The present invention relates to a kind of hyaluronic acid sodium gel, be specifically related to a kind of hyaluronic acid sodium gel injection for the osteoarthrosis chamber and preparation method thereof.
Background technology
(Osteoarthritis OA) is orthopaedics frequently-occurring disease and common disease clinically to osteoarthritis, and clinical serves as main performance with arthralgia, stiff and limitation of activity mainly, and the sickness rate of osteoarthritis increases progressively with the increase at age.World Health Organization's statistics, among the crowd more than 50 years old, the sickness rate of OA is 50%; The sickness rate of crowd more than 55 years old then rises to 80%.Report shows appropriate authority to the epidemiological survey in China some areas, more than 55 years old the crowd to suffer from the shower of gonarthritis be that the osteoarthritis prevalence of crowd more than 60%, 60 years old is 78.5%, the arthritic sickness rate of the old people of over-65s is 85%.
The joint comprises cartilage, synovial membrane and joint capsule.The basis of articular cartilage is chondrocyte and extracellular matrix, and wherein chondrocyte only accounts for 1%, and extracellular matrix accounts for 99%, and about 3% is glycosaminoglycans in the extracellular matrix.Hyaluronic acid (Hyaluronic Acid, HA) be a kind of macromolecular glycosaminoglycans (Glycosaminoglycan, GAG), extensively be present in the various tissues of organism, it extensively is present in human body institute in a organized way with the form of the outer mixture of histiocyte, and high concentration is present in knuckle synovia and the cartilage.
The existing joint disease that studies have shown that is closely related with the reduction of synovial fluid viscoelasticity, so viscoelasticity replacement therapy (J.Rheumatol such as Balazs, 1993,20; 3～9), namely by the method to intraarticular injection exogenous hyaluronic acid treatment osteoarthritis, obtained revolutionary progress from coming out.
The viscoelasticity that hyaluronic acid is special and rheological properties, particularly important to the meaning in joint.HA gives characteristic and the viscoelasticity of the synovial fluid non-Newtonian fluid mechanic of osteoarthrosis intracavity.When the joint was in low collision frequency, synovial fluid was viscosity solution, and synovial tissue, plane tissue, ligament and the collagen structure etc. in joint are exercised lubricating functions, reduces the friction between tissue; When bear a heavy burden in the joint; low molecule solute and moisture in the synovial fluid are pressed out in the cartilage matrix from synovial fluid; make synovial fluid be transformed into a gel state with several micron thickness; change elastomer into by fluid; serve as cushion pad in the joint space; alleviate the pressure that bear in the joint, to osteoarticular cartilage performance protective effect.
Hyaluronic acid is extremely important to the normal function of keeping synovial fluid as the important component part of knuckle synovia, makes articular cartilage and synovial fluid have better elastic.In osteoarthritis patient's knuckle synovia, the molecular weight of hyaluronate sodium, concentration and viscoelasticity all obviously reduce.By the hyaluronate sodium of the son amount of in articular cavity, directly awarding high marks, when exogenous hyaluronic acid sodium exists for a long time, rely on self repair of cartilaginous tissue in articular cavity, osteoarthritis is fully recovered.
But more existing products, as the Artz of Japanese Seikagaku Kogyo Co. Ltd., the Suplazyn of Canadian Bioniche company and the Sofast of Chinese Shandong Baushe ﹠ Lomb Fu Ruida, because metabolism is very fast, in articular cavity the time of staying shorter, need multiple injection.Multiple injection has not only increased the chance of irritant reaction and infection, also is burden to patient's physiology and psychology.
Hydroxyl in the HA molecule, carboxyl, N-acetamido can carry out chemical modification to it; HA derivant after the chemical modification has not only kept the original biocompatibility of HA and cell adhesion, compares with HA simultaneously to have prolonged life period in vivo.
In order to prolong hyaluronate sodium in the time of staying of articular cavity, existing cross-linked-hyaluronic acid or its sodium salt discovered overcome that natural hyaluronic acid easily disperses, degradation-labile defect, kept excellent biological compatibility, be present more satisfactory tissue filling material, crosslinked hyaluronic acid derivatives Restylan series and the Durolane series of products of glycidyl ether produced of Sweden Q-Med AB company for example.But have the residual of cross-linking agent in the above-mentioned product that comprises cross-linked-hyaluronic acid or its sodium salt, there is potential safety hazard in long term injections to human body.
In addition, Chinese patent literature CN 101502675A(application number 200810009190.8) discloses a kind of injection and contained the hyaluronic acid of macromolecule hydrogel or suspension of its salt and preparation method thereof, described macromolecular compound can be the cross-linked polysaccharides chemical compound, preferred 1, the crosslinked hyaluronic acid product of 4-butanediol diglycidyl ether (BDDE).During the preparation suspension, at first prepare the gel particle of macromolecular compound; With gel particle abundant swelling, balance in isosmotic solution, obtain macromolecular compound aqueous gel granule water-insoluble, abundant swelling in isosmotic solution then; At last previous step is obtained water-insoluble, in isosmotic solution the abundant isosmotic solution mix homogeneously of the macromolecular compound aqueous gel granule of swelling and HA or its salt and obtain suspension.
Above-mentioned document prepares macromolecule hydrogel for example during cross-linking hyaluronic acid sodium, be that hyaluronate sodium is mixed with 1% sodium hydroxide solution that contains crosslinking agent B DDE, in 50 ℃ of reactions 3 hours, behind the deionized water purification, pressurization makes gel pass through sieve and collects gel particle, the gel particle of collecting add wait ooze the PBS insulation after, cross 200 mesh sieves and obtain cross-linking sodium hyaluronate gel.
Chinese patent literature CN 101538377A(application number 200810034895.5) a kind of cross-linked hyaluronic acid gel and preparation method thereof is disclosed, earlier hyaluronic acid is mixed in alkaline substance solution, then to wherein adding cross-linking agent, reacted 2～5 hours down in 37 ℃～60 ℃, obtain using PBS buffer or deionized water soaking and washing gel behind the gel, after removing PBS buffer or deionized water, make cross-linked hyaluronic acid gel extrusion cavities by 450～550 μ m under pressure obtain microgranular gel; At last microgranular gel is mixed with hyaluronic acid and obtain described hyaluronic acid derivatives.
Above-mentioned two kinds of methods that prepare cross-linking hyaluronic acid sodium all are to carry out in the aqueous solution of alkali, have the hydrolysis problem of cross-linking hyaluronic acid sodium; Above-mentioned two kinds of methods are not all carried out the detection of residual BDDE in the cross-linking hyaluronic acid sodium in addition, can not confirm the residual condition of cross-linking agent in the gel; And behind the generation gel, even if with deionized water and PBS purification gel, the BDDE that part remains in the gel still can't remove.
Summary of the invention
Hyaluronic acid sodium gel injection that is used for the osteoarthrosis chamber that the time of staying is long and safety is higher when the purpose of this invention is to provide a kind of use the and preparation method thereof.
The technical scheme that realizes the object of the invention is a kind of preparation method of the hyaluronic acid sodium gel injection for the osteoarthrosis chamber, may further comprise the steps:
1. the preparation of cross-linking sodium hyaluronate gel:
A. hyaluronate sodium dry powder is dispersed in by obtaining hyaluronate sodium alkalescence suspension behind the sodium hydrate aqueous solution of 10 wt%～20 wt% and the mixed solution that acetone is formed, in hyaluronate sodium alkalescence suspension, add cross-linking agent 1 then, 4-butanediol diglycidyl ether BDDE, obtain reaction mass behind the mixing, thereby begin to generate the reaction of cross-linking hyaluronic acid sodium; Under the stirring reaction mass is finished in 5～8 hours afterreactions of 35 ℃～50 ℃ insulations, with the pH value to 7 of solidliquid mixture material behind the concentrated hydrochloric acid conditioned reaction; The concentration of hyaluronate sodium is 2wt%～5wt% in the wherein said reaction mass, and the mass ratio of cross-linking agent and hyaluronate sodium is (1: 1.3)～(1: 1.8).
B. the solidliquid mixture material of the pH=7 that step a is obtained filters to remove liquid, remaining material is lower than 2ppm with washing with acetone to BDDE content, remaining material comprises white powder and transparent gel, and it is the cross-linking hyaluronic acid sodium powder that the drying material in vacuum after will washing then obtains water-insoluble white dry powder.
C. the cross-linking hyaluronic acid sodium powder sieving separating that step b vacuum drying is obtained is collected the powder that sieves.
D. add deionized water in the powder of collecting to step c that sieves, make the abundant swelling of cross-linking hyaluronic acid sodium powder,, collect gel particle and obtain cross-linking sodium hyaluronate gel after 6～10 hours at 15 ℃～35 ℃ purification of room temperature.
E. add to wait in the gel of collecting to steps d and ooze the PBS buffer, after 6～10 hours, remove by filter the PBS buffer in 15 ℃～35 ℃ purification of room temperature, collecting gel particle, to obtain cross-linking sodium hyaluronate gel stand-by.
2. the configuration of hyaluronic acid sodium gel: other hyaluronate sodium dry powder is oozed in the PBS buffer fully swelling waiting, filter that to obtain hyaluronic acid sodium gel behind the unnecessary PBS solution stand-by.
3. mix and sterilize: the hyaluronic acid sodium gel mix homogeneously that 2. cross-linking sodium hyaluronate gel that will obtain according to step method 1. and step dispose, gel that mixing obtains sterilization back fill is oozed the hyaluronic acid sodium gel injection that obtains behind the PBS buffer standardize solution for the osteoarthrosis chamber with grade in the disposable syringe of prior sterilization.
1. among a, described mixed solution is when configuration for above-mentioned steps, and the volume ratio of described sodium hydrate aqueous solution and acetone is (2: 8)～(4: 6); Step 1. in the reactant liquor of a the concentration of hyaluronate sodium be 3 wt%～4wt%.
Above-mentioned steps 1. among the c cross-linking hyaluronic acid sodium powder sieving separating adopt 35 orders～45 purpose screen clothes; When 1. step added deionized water in the cross-linking hyaluronic acid sodium powder that sieves among the d, deionized water added back dry powder imbibition and forms gel, and the addition of deionized water guarantees the gel that there was not dry powder suction back to form fully; And change deionized water every 55min～80min in the purge process.
Above-mentioned steps 3. in, the mass ratio of cross-linking sodium hyaluronate gel and hyaluronic acid sodium gel is 8: 2 or 6: 4, after the gel sterilization that mixing obtains, get wherein 45g～50g fill in the disposable syringe of prior sterilization, ooze the PBS buffer and be settled to 6.0mL and obtain hyaluronic acid sodium gel injection for the osteoarthrosis chamber with waiting again.
A kind of gel injection that is prepared by the preparation method of the above-mentioned hyaluronic acid sodium gel injection that is used for the osteoarthrosis chamber.
A kind of second kind of preparation method of the hyaluronic acid sodium gel injection for the osteoarthrosis chamber is characterized in that may further comprise the steps:
1. the preparation of cross-linking hyaluronic acid sodium dry powder:
A. hyaluronate sodium dry powder is dispersed in by obtaining hyaluronate sodium alkalescence suspension behind the sodium hydrate aqueous solution of 10 wt%～20 wt% and the mixed solution that acetone is formed, in hyaluronate sodium alkalescence suspension, add cross-linking agent 1 then, 4-butanediol diglycidyl ether BDDE, obtain reaction mass behind the mixing, thereby begin to generate the reaction of cross-linking hyaluronic acid sodium; Under the stirring reaction mass is finished in 5～8 hours afterreactions of 35 ℃～50 ℃ insulations, with the pH value to 7 of solidliquid mixture material behind the concentrated hydrochloric acid conditioned reaction; The concentration of hyaluronate sodium is 2wt%～5wt% in the wherein said reaction mass, and the mass ratio of cross-linking agent and hyaluronate sodium is (1: 1.3)～(1: 1.8).
B. the solidliquid mixture material that step a is obtained filters to remove liquid, remaining material is lower than 2ppm with washing with acetone to BDDE content, remaining material comprises white powder and transparent gel, and it is the cross-linking hyaluronic acid sodium powder that the drying material in vacuum after will washing then obtains water-insoluble white dry powder.
C. the cross-linking hyaluronic acid sodium powder sieving separating that step b vacuum drying is obtained is collected the powder that sieves, and it is stand-by to obtain the cross-linking hyaluronic acid sodium powder.
2. it is stand-by that the cross-linking hyaluronic acid sodium powder that 1. step is obtained and other hyaluronate sodium dry powder mixing obtain mixed-powder.
3. swelling, purification and sterilization: add deionized water in the mixed-powder that 2. obtains to step, make cross-linking hyaluronic acid sodium powder and the abundant swelling of hyaluronate sodium powder,, collect gel particle and obtain mixed gel after 6～10 hours at 15 ℃～35 ℃ purification of room temperature.
Add in the above-mentioned mixed gel to wait and ooze the PBS buffer, in 15 ℃～35 ℃ purification of room temperature after 6～10 hours, remove by filter the PBS buffer, collect gel particle, sterilization, hyaluronic acid sodium gel fill after the sterilization obtains hyaluronic acid sodium gel injection for the osteoarthrosis chamber with PBS buffer standardize solution again in the disposable syringe of prior sterilization.
Above-mentioned steps 2. in, the mass ratio of cross-linking hyaluronic acid sodium powder and described hyaluronate sodium dry powder is 8: 2 or 6: 4.
Above-mentioned steps 3. in to after the gel particle sterilization of collecting, get wherein 45g～50g fill in the disposable syringe of prior sterilization, be settled to 6.0mL with the PBS buffer again and obtain hyaluronic acid sodium gel injection for the osteoarthrosis chamber.
1. among a, described mixed solution is when configuration for above-mentioned steps, and the volume ratio of described sodium hydrate aqueous solution and acetone is (2: 8)～(4: 6); Step 1. in the reactant liquor of a the concentration of hyaluronate sodium be 3wt%～4wt%; Step 1. among the c cross-linking hyaluronic acid sodium powder sieving separating adopt 35 orders～45 purpose screen clothes.
A kind of gel injection of second kind of preparation method preparation of aforesaid hyaluronic acid sodium gel injection for the osteoarthrosis chamber.
The present invention has positive effect:
(1) degradation speed of the hyaluronic acid sodium gel injection for the osteoarthrosis chamber of the present invention is slow, and the time of staying under the normal condition in articular cavity was 6 months; And cross-linking agent is residual in the gel is lower than 2ppm, after the injection of osteoarthrosis chamber, the toxic and side effects that produces because of cross-linking agent toxicity can not occur.
(2) the cross-linking sodium hyaluronate gel uniform particles in the gel injection of the present invention, apyrogeneity, aseptic, noresidue cross-linking agent, and cytotoxicity is low.
(3) the present invention is when the preparation cross-linking sodium hyaluronate gel, be that hyaluronate sodium and cross-linking agent are directly disperseed to mix to carry out cross-linking reaction in the aqueous slkali of acetone, alleviated the problem of the cross-linking hyaluronic acid sodium hydrolysis in the aqueous solution of alkali that makes in the prior art; In addition because crosslinking agent B DDE can be dissolved in the acetone, and acetone and water dissolve each other, so BDDE is dispersed in the reaction system, fully contacts with hyaluronate sodium, effectively improved cross-linking reaction speed and conversion ratio.
Material after cross-linking reaction finishes is removed the dry water-insoluble dry powder that forms of cross-linking agent final vacuum with organic solvent washing, because it mainly is to be dispersed in the acetone mixed solvent with the solid state powder shape that reaction finishes the back cross-linking hyaluronic acid sodium, have only a small amount of water absorption and swelling, can remove residual cross-linker as much as possible during therefore with washing with acetone.
(4) the present invention is when the preparation cross-linking sodium hyaluronate gel, and the water-insoluble dry powder that vacuum drying is obtained is crossed the separation of 40 mesh sieves earlier, and the powder that obtains sieving and unsifted powder are got the powder that sieves, and unsifted powder is thrown aside; And may wrap up impurity in the unsifted powder, therefore preparation method of the present invention has guaranteed the quality of final products.
(5) the present invention is when the preparation cross-linking sodium hyaluronate gel, gel particle is to be sieved earlier by the cross-linking hyaluronic acid sodium powder to sort out, be swelled into gel particle again, rather than obtain the gel of reunion in the traditional method earlier, the screen cloth by certain order number obtains under pressure again.
(6) the present invention has carried out the detection of rheology index to prepared gel injection, and the rheology index shows that gel of the present invention can be to osteoarticular cartilage performance protective effect.
(7) gel injection of the present invention is that the mean diameter with gel particle is that 150 μ m～cross-linking sodium hyaluronate gel of 300 μ m and the hyaluronic acid sodium gel of high molecular are made aseptic osteoarthrosis chamber injection after composite by different proportion, perhaps will cross the cross-linking hyaluronic acid sodium powder of 40 mesh sieves and hyaluronate sodium dry powder composite by different proportion after, make aseptic osteoarthrosis chamber injection after swelling, purification are composite; Compare with the injection of being made by the non-crosslinked hyaluronic acid sodium gel fully; cross-linking hyaluronic acid sodium in the injection of gained of the present invention can keep the longer time in synovial membrane and synovial membrane liquid; obtain excellent cartilage protection effect and permanent analgesic effect, and the hyaluronate sodium of non-crosslinked can disperse in synovial membrane liquid promptly to remain in the synovial membrane liquid with more low-level.Gel of the present invention is applicable to treatment gonalgia patient, the patient who is used for conventional non-drug therapy and uses antipyretic analgesic to cure.
Description of drawings
Fig. 1 is the structural formula of hyaluronate sodium;
Fig. 2 connects sketch map for the cancellated chemical bond of the cross-linking hyaluronic acid sodium of the present invention's preparation, and wherein A is-CH
2-CH(OH)-CH
2-O-CH
2-CH
2--CH
2-CH
2-O-CH
2-CH(OH)-CH
2-;
The particle size distribution figure of the gel that Fig. 3 obtains for the cross-linking hyaluronic acid sodium powder swelling purification of mistake 40 mesh sieves of embodiment 1 preparation;
The GC-MS of residual cross-linker BDDE detects spectrogram in the cross-linking sodium hyaluronate gel that Fig. 4 makes for embodiment 1;
Fig. 5 is apparent viscosity and the time relation curve of the hyaluronic acid sodium gel injection that is used for the osteoarthrosis chamber of embodiment 1 preparation;
Fig. 6 is the viscoelasticity curve chart of the hyaluronic acid sodium gel injection that is used for the osteoarthrosis chamber of embodiment 1 preparation;
Fig. 7 is apparent viscosity and the time relation curve of the hyaluronic acid sodium gel injection that is used for the osteoarthrosis chamber of embodiment 3 preparations;
Fig. 8 is the viscoelasticity curve chart of the hyaluronic acid sodium gel injection that is used for the osteoarthrosis chamber of embodiment 3 preparations.
The specific embodiment
(embodiment 1)
The present embodiment agents useful for same is pharmaceutical grade.
The preparation method of the hyaluronic acid sodium gel injection that is used for the osteoarthrosis chamber of present embodiment may further comprise the steps:
1. the preparation of cross-linking sodium hyaluronate gel.
A. add the mixed solution that sodium hydrate aqueous solution and the acetone by 15wt% of 500mL are formed in hyaluronate sodium (structural formula as shown in Figure 1) dry powder of the white of constant weight, perhaps the hyaluronate sodium dry powder of the white of adding constant weight in the mixed solution of forming to sodium hydrate aqueous solution and the acetone by 15wt% of 500mL; The mean molecule quantity of described hyaluronate sodium is 500,000～1,600,000 dalton (being 1,500,000 dalton in the present embodiment); Stirring makes hyaluronate sodium evenly disperse the back to obtain hyaluronate sodium alkalescence suspension, and namely hyaluronate sodium is partially dissolved in the mixed solution of sodium hydrate aqueous solution and acetone composition, also has part to exist with solid-state form; Add the cross-linking agent 1 of 11.1mL then in the hyaluronate sodium alkalescence suspension, 4-butanediol diglycidyl ether (BDDE) obtains reaction mass behind the mixing, thereby begins to generate the reaction of cross-linking hyaluronic acid sodium.The concrete numerical control of the weight of the hyaluronate sodium dry powder of above-mentioned white is in described reaction mass, and the concentration of hyaluronate sodium is 2wt%～5wt%, and in the present embodiment, the weight of the hyaluronate sodium dry powder of white is 20g.
Under stirring reaction mass is finished in 5 hours afterreactions of 40 ℃ of insulations, the pH value to 7 of solidliquid mixture material behind the concentrated hydrochloric acid conditioned reaction of adding 37wt%, the cancellated chemical bond of cross-linking hyaluronic acid sodium connect signal and see Fig. 2.The mixed solution of above-mentioned sodium hydrate aqueous solution and acetone is that the sodium hydrate aqueous solution of 15wt% and the acetone volume ratio mixing according to (2: 8)～(4: 6) is obtained, and is 3: 7 in the present embodiment.(CAS number: 9067-32-7) dry powder is for from the hyaluronate sodium dry powder of cockscomb or the hyaluronate sodium dry powder that is obtained by bacterial fermentation for described hyaluronate sodium.
B. the solidliquid mixture material with step a reaction back pH=7 filters to remove liquid, comprise white cross-linking hyaluronic acid sodium powder and transparent cross-linking sodium hyaluronate gel in the remaining material, leftover materials detect BDDE content with washing with acetone to gas chromatograph-mass spectrometer (GC-MS) (GC-MS) and are lower than 2ppm; It is the cross-linking hyaluronic acid sodium powder that drying material in vacuum after will washing then obtains water-insoluble white dry powder.Used gas chromatograph-mass spectrometer (GC-MS) is day QP-2010 type gas chromatograph-mass spectrometer (GC-MS) of island proper Tianjin company.
C. the cross-linking hyaluronic acid sodium powder that step b vacuum drying is obtained is crossed the separation of 40 mesh sieves, collects the powder that sieves, and unsifted powder is given it up owing to wherein may comprise the impurity with viscosity.
D. add the capacity deionized water in the cross-linking hyaluronic acid sodium powder of mistake 40 mesh sieves of collecting to step c, make the abundant swelling of cross-linking hyaluronic acid sodium, at 25 ℃ of purification of room temperature after 8 hours, collect gel particle and obtain cross-linking sodium hyaluronate gel, changed deionized water in the purge process every 1 hour.
The dry powder imbibition forms gel after adding deionized water, and the addition of deionized water should guarantee there be not fully the gel of dry powder suction back formation.
E. the gel of collecting to the steps d grade that adds 3 times of gel volumes is oozed the PBS buffer (NaH that every 1000mL contains
2PO
42H
2O is 90mg, Na
2HPO
412H
2O is 1.12g, and NaCl is 17g, and all the other are water), after 8 hours, remove by filter the PBS buffer in 25 ℃ of purification of room temperature, collect gel particle and obtain cross-linking sodium hyaluronate gel, gel is stand-by after 15 minutes in 120 ℃ of high-temperature sterilizations.
In order to understand the character of the cross-linking sodium hyaluronate gel of preparation according to the method described above, in accordance with the following methods gel is detected:
1, the detection of hyaluronate sodium (SH) content in the gel.
Getting the gel after the 0.5g step e sterilization, is in the sulfuric acid solution of 0.5mol/L with its concentration that adds 10mL, and the boiling water bath hydrolysis is after 15 minutes, and adding water to volume in the solution after the hydrolysis is 100mL.Measure glucuronic acid content with the carbazole method, then multiplying factor 2.07 2.05wt%, be the content of the hyaluronate sodium SH in the gel of present embodiment preparation.
2, the granularity Detection of gel particle in the gel.Granularity Detection is used the Mastersizer 2000 type particle size analyzers of Britain Malvern company.
Gel 0.1g after the step e sterilization is sent into particle size analyzer detect, the particle size distribution figure that records is seen Fig. 3, and the mean diameter of gel particle is 206.172 μ m, and the gel particle that makes is more even.
3, vitro cytotoxicity detects.According to the GB/T16886 national standard, cross-linking hyaluronic acid sodium carries out the vitro cytotoxicity test as national three class medical apparatus and instruments.
Earlier cross-linking hyaluronic acid sodium is mixed by 0.2g/mL with the RPMI1640 culture fluid, place 37 ℃, lixiviate is 72 hours in the 5wt% carbon dioxide incubator, with 0.22 μ m filtering with microporous membrane degerming, obtains lixiviating solution.
L929 cell suspending liquid with 1*105/mL is inoculated in 96 porocyte culture plates then, places 37 ℃ of CO2 gas incubator to cultivate 24 hours; After treating the cell attachment growth, remove supernatant, be divided into two groups of blank group and experimental grouies, matched group adds the RPMI1640 culture fluid, and experimental group exchanges with the RPMI1640 culture fluid that contains the above-mentioned lixiviating solution of 50wt%.Place 37 ℃ of CO2 gas incubator to continue to cultivate respectively, took out after 2 days, the every hole of culture plate adds MTT solution (5mg/mL) 20 μ L, continues to cultivate 4 hours in 37 ℃, stops cultivating.The careful suction abandoned culture supernatant in the hole, and every hole adds 200 μ L DMSO, behind the 10 minutes mixings of vibrating, measures its absorbance respectively with enzyme-linked immunosorbent assay instrument under 630nm.
Calculate the relative appreciation rate of cell (RCR), RCR(%)=(experimental group mean light absorbency value/blank group mean light absorbency value) * 100% according to following formula.
The relative appreciation rate of cell and cytotoxicity classification relationship are as follows: RCR is not less than 100%, and cytotoxicity is classified as 0 grade; RCR is 75-99%, and cytotoxicity is classified as 1 grade; RCR is 50-74%, and cytotoxicity is classified as 2 grades; RCR is 25-49%, and cytotoxicity is classified as 3 grades; RCR is 1-24%, and cytotoxicity is classified as 4 grades; RCR is 0%, and cytotoxicity is classified as 5 grades.
Cross-linking sodium hyaluronate gel after the present embodiment step e sterilization according to the method described above, RCR is 83.69%, cytotoxicity is low.
4, anti-enzyme detection.Get 0.5g cross-linking sodium hyaluronate gel to be measured, to add pH after it adds the hyaluronic acid enzymatic solution that 1.25mL concentration is 10U/mL be 7.2 PBS to volume be 2.5mL, in 37 ℃ of enzymolysis 24 hours; With centrifuge with 10000rpm centrifugal 40 minutes, get supernatant, according to the centrifugalize twice again of above-mentioned centrifugal method, all get supernatant at every turn, merge supernatant.Adopt improvement click flight of steps leading to a palace hall development process (list of references: Bitter .T, Muir H.M, (1962) A modified uronic acid carbarbazole reation .Anal.Biochem.4,330-333.) the mensuration glucuronic acid content, multiply by 2.07 back conversions and be the hyaluronic acid sodium content, as a value, be that 2.05wt% is the b value with hyaluronic acid sodium content c in the gel, calculate a/b.
Cross-linking sodium hyaluronate gel after the present embodiment step e sterilization according to the method described above, a/b=0.27.
5, the detection of content of beary metal in the gel.With reference to the method for 2005 editions two appendix of Chinese Pharmacopoeia, get gel 1g to be measured, blazing to carbonization 60 ℃ of oven dry, add 0.5mL sulphuric acid heating evaporate to dryness, add 0.5mL nitric acid heating evaporate to dryness again, make ashing 500 ℃～600 ℃ heating; Add 2mL hydrochloric acid then, behind the water bath method, add the 15mL distilled water, be adjusted to neutrality with the ammonia of 4 wt %, add 2mL pH and be 3.5 acetate buffer, be diluted to 25mL after the dissolving; After reference substance is got above-mentioned agents useful for same evaporate to dryness simultaneously, add equivalent acetate buffer solution and distilled water, add the plumbous test solution of an amount of standard, be diluted to 25mL, this moment, lead content was 10ppm; Carry out audit by comparison after adding the colour developing of thioacetamide test solution respectively.
Cross-linking sodium hyaluronate gel after the present embodiment step e sterilization according to the method described above, the content of beary metal of gel is less than 10ppm.
In order to understand the definite content of each heavy metal in the gel, further with inductively coupled plasma spectrum generator gel is detected, checkout equipment is day ICP-7510 of island proper Tianjin company, 20 ℃ of the temperature of testing environment, humidity 50%.Testing result is as follows:
6, the detection of cross-linking agent residual quantity in the gel.
The detection of cross-linking agent residual quantity adopts GC-MS to detect in the gel, and adopting instrument is day QP-2010 model gas chromatograph-mass spectrometer (GC-MS) of island proper Tianjin company.
The at first blank feasibility that reclaims this method of checking, getting the BDDE sample 5mL of 10ppm, is 15ppm to the concentration that wherein adds BDDE to BDDE, carries out 6 times through GC-MS and detects, the content for the treatment of BDDE in the test sample is 14.9 ± 0.1ppm, and it detects precision and reaches requirement.
Test sample is treated in configuration then, gets cross-linking sodium hyaluronate gel 1g to be measured, adds water 1mL, uses the analytical pure dichloromethane extraction again 2 times, the each consumption 10mL of dichloromethane; Combining extraction liquid steams after the extract drying and removes dichloromethane until obtaining the dichloromethane solution that 0.2mL contains BDDE, detects through GC-MS then.
Cross-linking sodium hyaluronate gel after the present embodiment step e sterilization according to the method described above, the GC spectrogram that GC-MS detects is seen Fig. 4, the content that records BDDE in the gel is 0.7239ppm.
2. the configuration of hyaluronic acid sodium gel.
Be averaged molecular weight and be 1,500,000 daltonian hyaluronate sodiums white dry powder 20g, ooze PBS solution 200mL to wherein adding to wait, make the abundant swelling of hyaluronate sodium dry powder, obtain hyaluronic acid sodium gel after filtering out unnecessary PBS solution; Described grade is oozed the NaH that every 1000mL contains in the PBS solution
2PO
42H
2O is 90mg, Na
2HPO
412H
2O is 1.12g, and NaCl is 17g, and all the other are water.
3. mix and sterilize.
Behind the hyaluronic acid sodium gel 9.6g mix homogeneously that 2. the cross-linking sodium hyaluronate gel 38.4g that will obtain according to step method 1. and step dispose (both mass ratioes are 8: 2), at 120 ℃ of flowing steam sterilizations (also i.e. sterilization) after 20 minutes, its fill in the disposable syringe of prior sterilization, is oozed the PBS buffer and is settled to 6.0mL and obtains hyaluronic acid sodium gel injection for the osteoarthrosis chamber with waiting again.
In order to understand the character of the hyaluronic acid sodium gel injection that is used for the osteoarthrosis chamber that makes, carry out vitro cytotoxicity detection, anti-enzyme detection, the detection of cross-linking agent residual quantity and rheology index and detect.
(1) vitro cytotoxicity detects.The method that the vitro cytotoxicity of the hyaluronic acid sodium gel injection that is used for the osteoarthrosis chamber that 3. step makes detects is identical with the detection method of the cross-linking sodium hyaluronate gel that 1. step makes, and recording the relative appreciation rate RCR of cell is 82.3%, and cytotoxicity is low.
(2) anti-enzyme detection.The method of the anti-enzyme detection of the hyaluronic acid sodium gel injection that is used for the osteoarthrosis chamber that 3. step makes is identical with the detection method of the cross-linking sodium hyaluronate gel that 1. step makes, and records a/b=0.39.
(3) detection of content of beary metal in the gel.The content of beary metal of the hyaluronic acid sodium gel injection that is used for the osteoarthrosis chamber that 3. detection method of the content of beary metal of the cross-linking sodium hyaluronate gel that 1. makes according to step, step make is less than 10ppm.
(4) detection of cross-linking agent residual quantity in the gel.The content of the hyaluronic acid sodium gel injection crosslinking agent B DDE that is used for the osteoarthrosis chamber that 3. detection method of the cross-linking agent residual quantity of the cross-linking sodium hyaluronate gel that 1. makes according to step, step make is 0.5790 ppm.
(5) detection of gel injection rheology index.
Britain Malvern CVO100 dynamic shear rheometer DSR is adopted in the detection of the rheology index of cross-linking sodium hyaluronate gel injection to be measured.
The setting measurement temperature is 25, under the VISCOMETRY pattern, selects rate controlled, the setting shear rate is 2.0001/s, time delay 20s, the time of integration 5s, adopt and count 15, obtain the viscosity number of cross-linking sodium hyaluronate gel injection, concrete measurement result is seen Fig. 5 and following table 1.
The english nouns correspondence of first row is Chinese as follows in the table 1: Time-time, Temperature-temperature, Target Shear Rate-target shear rate, Shear Rate-shear rate, Percentage Deviation-percentage deviation, Shear Stress-shear stress, Viscosity-viscosity.
Table 1
By table 1 testing result as can be known, at 25 ℃, shear rate is 2s
-1The time, the dynamic viscosity of cross-linking sodium hyaluronate gel injection is about 60000 mPas, is far longer than 45000mPas.
Same setting measurement temperature is 25, under the OSCILLATION pattern, select frequency scanning, rate of scanning is from 0.01Hz to 10Hz, the concrete measurement result of every rheology index of cross-linking sodium hyaluronate gel injection sees the following form 2 and table 3, and the viscoelasticity curve chart is seen Fig. 6.
The english nouns correspondence of first row is Chinese as follows in table 2, the table 3: Time-time, Temperature-temperature, Frequency-frequency, Phase Angle-phase angle, Complex Modulus-complex modulus, Elastic Modulus-elastic modelling quantity, Viscous Modulus-viscous modulus, Complex Viscosity-complex viscosity, Shear Stress-shear stress, Strain-strain.
Table 2
Table 3
By table 2, table 3 and Fig. 6 as can be known, when frequency of oscillation was 0.01Hz, the complex viscosity value of cross-linking sodium hyaluronate gel was 2922.5 Pas, greater than 1000Pas; When frequency of oscillation was 10Hz, the complex viscosity value of cross-linking sodium hyaluronate gel was 8.1907Pas, greater than 3.5Pas.
(embodiment 2)
The preparation method of the hyaluronic acid sodium gel injection that is used for the osteoarthrosis chamber of present embodiment may further comprise the steps:
1. the preparation of cross-linking hyaluronic acid sodium dry powder.
A. add the mixed solution that sodium hydrate aqueous solution and the acetone by 15wt% of 500mL are formed in hyaluronate sodium (structural formula as shown in Figure 1) dry powder of the white of constant weight, perhaps the hyaluronate sodium dry powder of the white of adding constant weight in the mixed solution of forming to sodium hydrate aqueous solution and the acetone by 15wt% of 500mL; The mean molecule quantity of described hyaluronate sodium is 500,000～1,600,000 dalton (being 1,500,000 dalton in the present embodiment); Stirring makes hyaluronate sodium evenly disperse the back to obtain hyaluronate sodium alkalescence suspension; Add the cross-linking agent 1 of 11.1mL then in the hyaluronate sodium alkalescence suspension, 4-butanediol diglycidyl ether (BDDE) obtains reaction mass behind the mixing, thereby begins to generate the reaction of cross-linking hyaluronic acid sodium.The concrete numerical control of the weight of the hyaluronate sodium dry powder of above-mentioned white is in described reaction mass, and the concentration of hyaluronate sodium is 2wt%～5wt%, and in the present embodiment, the weight of the hyaluronate sodium dry powder of white is 20g.
Under stirring, reaction mass is finished the pH value to 7 of solidliquid mixture material behind the concentrated hydrochloric acid conditioned reaction of adding 37wt% in 5 hours afterreactions of 40 ℃ of insulations.The mixed solution that above-mentioned sodium hydrate aqueous solution and acetone are formed is that 15% sodium hydrate aqueous solution and the acetone volume ratio mixing according to (2: 8)～(4: 6) is obtained, and is 3: 7 in the present embodiment.Described hyaluronate sodium dry powder is for from the hyaluronate sodium dry powder of cockscomb or the hyaluronate sodium dry powder that is obtained by bacterial fermentation.
B. the solidliquid mixture material with the reacted pH=7 of step a filters to remove liquid, comprise white cross-linking hyaluronic acid sodium powder and transparent cross-linking sodium hyaluronate gel in the remaining material, leftover materials detect BDDE content with washing with acetone to gas chromatograph-mass spectrometer (GC-MS) (GC-MS) and are lower than 2ppm; It is the cross-linking hyaluronic acid sodium powder that drying material in vacuum after will washing then obtains water-insoluble white dry powder.
Used gas chromatograph-mass spectrometer (GC-MS) is day QP-2010 type gas chromatograph-mass spectrometer (GC-MS) of island proper Tianjin company.
C. the cross-linking hyaluronic acid sodium powder that step b vacuum drying is obtained is crossed the separation of 40 mesh sieves, and it is stand-by to collect the powder that sieves, and unsifted powder is given it up owing to wherein may comprise impurity.
2. to be according to weight ratio that 8: 2 mixings obtain mixed-powder stand-by for the cross-linking hyaluronic acid sodium powder that will obtain according to step method 1. and other hyaluronate sodium powder.
3. swelling, purification and sterilization: add deionized water in the mixed-powder that 2. obtains to step, make cross-linking hyaluronic acid sodium powder and the abundant swelling of hyaluronate sodium powder,, collect gel particle and obtain mixed gel after 6～10 hours at 15 ℃～35 ℃ purification of room temperature.
Add in the above-mentioned mixed gel to wait and ooze the PBS buffer, in 15 ℃～35 ℃ purification of room temperature after 6～10 hours, remove by filter the PBS buffer, collect gel particle, get wherein 48g fill after the sterilization in the disposable syringe of prior sterilization, be settled to 6.0mL with the PBS buffer again and obtain hyaluronic acid sodium gel injection for the osteoarthrosis chamber.
Hyaluronic acid sodium gel injection to the present embodiment preparation carries out vitro cytotoxicity detection, anti-enzyme detection and the detection of rheology index.
(1) vitro cytotoxicity detects.According to the method that the vitro cytotoxicity of embodiment 1 detects, recording RCR is 82.5%, and cytotoxicity is low.
(2) anti-enzyme detection.Method according to the anti-enzyme detection of embodiment 1 records a/b=0.39.
(3) detection of content of beary metal in the gel.According to the detection method of the content of beary metal of embodiment 1, the content of beary metal of the hyaluronic acid sodium gel injection of present embodiment is less than 10ppm.
(4) detection of gel injection rheology index.
According to detecting instrument and the parameter of embodiment 1, at 25 ℃, shear rate is 2s
-1The time, the dynamic viscosity of the cross-linking sodium hyaluronate gel injection of present embodiment is about 60000 mPas, is far longer than 45000mPas.
When frequency of oscillation was 0.01Hz, the complex viscosity value of cross-linking sodium hyaluronate gel was 2921.8 Pas, greater than 1000Pas; When frequency of oscillation was 10Hz, the complex viscosity value of cross-linking sodium hyaluronate gel was 8.1527 Pas, greater than 3.5Pas.
(embodiment 3)
All the other are identical with embodiment 1 for the preparation method of the hyaluronic acid sodium gel injection that is used for the osteoarthrosis chamber of present embodiment, and difference is:
Step 3. in, the cross-linking sodium hyaluronate gel that obtains according to step method 1. of taking is 28.8g, the hyaluronic acid sodium gel according to step method configuration 2. of taking is 19.2g.Both mass ratioes are 6: 4.
Hyaluronic acid sodium gel injection to the present embodiment preparation carries out vitro cytotoxicity detection, anti-enzyme detection and the detection of rheology index.
(1) vitro cytotoxicity detects.According to the method that the vitro cytotoxicity of embodiment 1 detects, recording RCR is 87.5%, and cytotoxicity is low.
(2) anti-enzyme detection.Method according to the anti-enzyme detection of embodiment 1 records a/b=0.46.
(3) detection of content of beary metal in the gel.According to the detection method of the content of beary metal of embodiment 1, the content of beary metal of the hyaluronic acid sodium gel injection of present embodiment is less than 10ppm.
(4) detection of gel injection rheology index.
The setting measurement temperature is 25, under the VISCOMETRY pattern, selects rate controlled, the setting shear rate is 2.0001/s, time delay 20s, the time of integration 5s, adopt and count 15, obtain the viscosity number of cross-linking sodium hyaluronate gel injection, concrete measurement result is seen Fig. 7 and following table 4.
The english nouns correspondence of first row is Chinese as follows in the table 4: Time-time, Temperature-temperature, Target Shear Rate-target shear rate, Shear Rate-shear rate, Percentage Deviation-percentage deviation, Shear Stress-shear stress, Viscosity-viscosity.
By table 4 testing result as can be known, at 25 ℃, shear rate is 2s
-1The time, the dynamic viscosity of cross-linking sodium hyaluronate gel injection is about 42000mPas.
Table 4
Same setting measurement temperature is 25, under the OSCILLATION pattern, select frequency scanning, rate of scanning is from 0.01Hz to 10Hz, the concrete measurement result of every rheology index of cross-linking sodium hyaluronate gel injection sees the following form 5 and table 6, and the viscoelasticity curve chart is seen Fig. 8.
The english nouns correspondence of first row is Chinese as follows in table 5 and the table 6: Time-time, Temperature-temperature, Frequency-frequency, Phase Angle-phase angle, Complex Modulus-complex modulus, Elastic Modulus-elastic modelling quantity, Viscous Modulus-viscous modulus, Complex Viscosity-complex viscosity, Shear Stress-shear stress, Strain-strain.
By table 5, table 6 and Fig. 8 as can be known, when frequency of oscillation was 0.01Hz, the complex viscosity value of cross-linking sodium hyaluronate gel was 1347.4 Pas, greater than 1000Pas; When frequency of oscillation was 10Hz, the complex viscosity value of cross-linking sodium hyaluronate gel was 5.5227Pas, greater than 3.5Pas.
Table 5
Table 6
(embodiment 4)
All the other are identical with embodiment 2 for the preparation method of the hyaluronic acid sodium gel injection that is used for the osteoarthrosis chamber of present embodiment, and difference is:
Hyaluronic acid sodium gel injection to the present embodiment preparation carries out vitro cytotoxicity detection, anti-enzyme detection and the detection of rheology index.
(1) vitro cytotoxicity detects.According to the method that the vitro cytotoxicity of embodiment 1 detects, recording RCR is 87.5%, and cytotoxicity is low.
(2) anti-enzyme detection.Method according to the anti-enzyme detection of embodiment 1 records a/b=0.46.
(3) detection of content of beary metal in the gel.According to the detection method of the content of beary metal of embodiment 1, the content of beary metal of the hyaluronic acid sodium gel injection of present embodiment is less than 10ppm.
(4) detection of gel injection rheology index.
According to detecting instrument and the parameter of embodiment 1, at 25 ℃, shear rate is 2s
-1The time, the dynamic viscosity of the cross-linking sodium hyaluronate gel injection of present embodiment is about 42000mPas, is far longer than 30000mPas.
When frequency of oscillation was 0.01Hz, the complex viscosity value of cross-linking sodium hyaluronate gel was 1347.2Pas, greater than 1000Pas; When frequency of oscillation was 10Hz, the complex viscosity value of cross-linking sodium hyaluronate gel was 5.5236Pas, greater than 3.5Pas.
(embodiment 5)
All the other are identical with embodiment 1 for the preparation method of the hyaluronic acid sodium gel injection that is used for the osteoarthrosis chamber of present embodiment, difference is: step 3. in, the cross-linking sodium hyaluronate gel of making according to step method 1. of taking is 40g, and the hyaluronic acid sodium gel according to step method configuration 2. of taking is 5g.The hyaluronic acid sodium gel that is used for the osteoarthrosis chamber of present embodiment preparation is according to the anti-enzyme detection method of embodiment 1, a/b=0.30.Therefore, the gel of present embodiment preparation is strong than the anti-enzyme of the gel of embodiment 1.
(embodiment 6)
All the other are identical with embodiment 1 for the preparation method of the hyaluronic acid sodium gel injection that is used for the osteoarthrosis chamber of present embodiment, difference is: step 3. in, the cross-linking sodium hyaluronate gel of making according to step method 1. of taking is 25g, and the hyaluronic acid sodium gel according to step method configuration 2. of taking is 25g.The hyaluronic acid sodium gel that is used for the osteoarthrosis chamber of present embodiment preparation is according to the anti-enzyme detection method of embodiment 1, a/b=0.56.Therefore, comparing embodiment 1, embodiment 5 and embodiment 6, along with the content of the hyaluronic acid sodium gel injection cross-linking hyaluronic acid sodium that is used for the osteoarthrosis chamber is more high, the anti-enzyme of the gel of gained is more strong.
(embodiment 7)
All the other are identical with embodiment 2 for the preparation method of the hyaluronic acid sodium gel injection that is used for the osteoarthrosis chamber of present embodiment, difference is: step 2. in, the weight ratio of the cross-linking hyaluronic acid sodium powder that obtains according to step method 1. of taking and other hyaluronate sodium powder is 1: 1.
Step 3. in, the gel particle after the sterilization of fill in the disposable syringe of prior sterilization is 50g.
The hyaluronic acid sodium gel that is used for the osteoarthrosis chamber of present embodiment preparation is according to the anti-enzyme detection method of embodiment 1, a/b=0.55.
Compare with the gel injection of embodiment 2 preparations, a little less than the anti-enzyme of gel than the gel of embodiment 2 of present embodiment preparation, the time of staying in vivo is short.
(embodiment 8)
All the other are identical with embodiment 2 for the preparation method of the hyaluronic acid sodium gel injection that is used for the osteoarthrosis chamber of present embodiment, difference is: step 2. in, the weight ratio of the cross-linking hyaluronic acid sodium powder that obtains according to step method 1. of taking and other hyaluronate sodium powder is 8: 1.
The hyaluronic acid sodium gel that is used for the osteoarthrosis chamber of present embodiment preparation is according to the anti-enzyme detection method of embodiment 1, a/b=0.31.Therefore, with the gel phase ratio of embodiment 2 preparations, the gel of present embodiment preparation is strong than the anti-enzyme of the gel of embodiment 2, and the time of staying in vivo is long.
Claims (10)
1. preparation method that is used for the hyaluronic acid sodium gel injection in osteoarthrosis chamber is characterized in that may further comprise the steps:
1. the preparation of cross-linking sodium hyaluronate gel:
A. obtain hyaluronate sodium alkalescence suspension after hyaluronate sodium dry powder being dispersed in the mixed solution of being formed by sodium hydrate aqueous solution and the acetone of 10wt%～20wt%, described mixed solution is when preparation, and the volume ratio of described sodium hydrate aqueous solution and acetone is (2: 8)～(4: 6); Add cross-linking agent 1 then in hyaluronate sodium alkalescence suspension, 4-butanediol diglycidyl ether BDDE obtains reaction mass behind the mixing, thereby begins to generate the reaction of cross-linking hyaluronic acid sodium; Under the stirring reaction mass is finished in 5～8 hours afterreactions of 35 ℃～50 ℃ insulations, with the pH value to 7 of solidliquid mixture material behind the concentrated hydrochloric acid conditioned reaction; The concentration of hyaluronate sodium is 2wt%～5wt% in the wherein said reaction mass, and the mass ratio of cross-linking agent and hyaluronate sodium is (1: 1.3)～(1: 1.8);
B. the solidliquid mixture material of the pH=7 that step a is obtained filters to remove liquid, remaining material is lower than 2ppm with washing with acetone to BDDE content, remaining material comprises white powder and transparent gel, and it is the cross-linking hyaluronic acid sodium powder that the drying material in vacuum after will washing then obtains water-insoluble white dry powder;
C. the cross-linking hyaluronic acid sodium powder sieving separating that step b vacuum drying is obtained is collected the powder that sieves;
D. add deionized water in the powder of collecting to step c that sieves, make the abundant swelling of cross-linking hyaluronic acid sodium powder,, collect gel particle and obtain cross-linking sodium hyaluronate gel after 6～10 hours at 15 ℃～35 ℃ purification of room temperature;
E. add to wait in the gel of collecting to steps d and ooze the PBS buffer, after 6～10 hours, remove by filter the PBS buffer in 15 ℃～35 ℃ purification of room temperature, collecting gel particle, to obtain cross-linking sodium hyaluronate gel stand-by;
2. the preparation of hyaluronic acid sodium gel: other hyaluronate sodium dry powder is oozed in the PBS buffer fully swelling waiting, filter that to obtain hyaluronic acid sodium gel behind the unnecessary PBS solution stand-by;
3. mix and sterilize: the hyaluronic acid sodium gel mix homogeneously that 2. cross-linking sodium hyaluronate gel that will obtain according to step method 1. and step are prepared, gel that mixing obtains sterilization back fill is oozed the hyaluronic acid sodium gel injection that obtains behind the PBS buffer standardize solution for the osteoarthrosis chamber with grade in the disposable syringe of prior sterilization.
2. the preparation method of the hyaluronic acid sodium gel injection for the osteoarthrosis chamber according to claim 1 is characterized in that: step 1. in the reactant liquor of a the concentration of hyaluronate sodium be 3wt%～4wt%.
3. the preparation method of the hyaluronic acid sodium gel injection for the osteoarthrosis chamber according to claim 1 is characterized in that: step 1. among the c cross-linking hyaluronic acid sodium powder sieving separating adopt 35 orders～45 purpose screen clothes; When 1. step added deionized water in the cross-linking hyaluronic acid sodium powder that sieves among the d, deionized water added back dry powder imbibition and forms gel, and the addition of deionized water guarantees the gel that there was not dry powder suction back to form fully; And change deionized water every 55min～80min in the purge process.
4. the preparation method of the hyaluronic acid sodium gel injection for the osteoarthrosis chamber according to claim 1, it is characterized in that: step 3. in, the mass ratio of cross-linking sodium hyaluronate gel and hyaluronic acid sodium gel is 8: 2 or 6: 4, after the gel sterilization that mixing obtains, get wherein 45g～50g fill in the disposable syringe of prior sterilization, ooze the PBS buffer and be settled to 6.0mL and obtain hyaluronic acid sodium gel injection for the osteoarthrosis chamber with waiting again.
5. gel injection by the preparation method preparation of the described hyaluronic acid sodium gel injection for the osteoarthrosis chamber of claim 1.
6. preparation method that is used for the hyaluronic acid sodium gel injection in osteoarthrosis chamber is characterized in that may further comprise the steps:
1. the preparation of cross-linking hyaluronic acid sodium dry powder:
A. obtain hyaluronate sodium alkalescence suspension after hyaluronate sodium dry powder being dispersed in the mixed solution of being formed by sodium hydrate aqueous solution and the acetone of 10wt%～20wt%, described mixed solution is when preparation, and the volume ratio of described sodium hydrate aqueous solution and acetone is (2: 8)～(4: 6); Add cross-linking agent 1 then in hyaluronate sodium alkalescence suspension, 4-butanediol diglycidyl ether BDDE obtains reaction mass behind the mixing, thereby begins to generate the reaction of cross-linking hyaluronic acid sodium; Under the stirring reaction mass is finished in 5～8 hours afterreactions of 35 ℃～50 ℃ insulations, with the pH value to 7 of solidliquid mixture material behind the concentrated hydrochloric acid conditioned reaction; The concentration of hyaluronate sodium is 2wt%～5wt% in the wherein said reaction mass, and the mass ratio of cross-linking agent and hyaluronate sodium is (1: 1.3)～(1: 1.8);
B. the solidliquid mixture material of the pH=7 that step a is obtained filters to remove liquid, remaining material is lower than 2ppm with washing with acetone to BDDE content, remaining material comprises white powder and transparent gel, and it is the cross-linking hyaluronic acid sodium powder that the drying material in vacuum after will washing then obtains water-insoluble white dry powder;
C. the cross-linking hyaluronic acid sodium powder sieving separating that step b vacuum drying is obtained is collected the powder that sieves, and it is stand-by to obtain the cross-linking hyaluronic acid sodium powder;
2. it is stand-by that the cross-linking hyaluronic acid sodium powder that 1. step is obtained and other hyaluronate sodium dry powder mixing obtain mixed-powder;
3. swelling, purification and sterilization: add deionized water in the mixed-powder that 2. obtains to step, make cross-linking hyaluronic acid sodium powder and the abundant swelling of hyaluronate sodium powder,, collect gel particle and obtain mixed gel after 6～10 hours at 15 ℃～35 ℃ purification of room temperature;
Add in the above-mentioned mixed gel to wait and ooze the PBS buffer, in 15 ℃～35 ℃ purification of room temperature after 6～10 hours, remove by filter the PBS buffer, collect gel particle, sterilization, hyaluronic acid sodium gel fill after the sterilization obtains hyaluronic acid sodium gel injection for the osteoarthrosis chamber with PBS buffer standardize solution again in the disposable syringe of prior sterilization.
7. the preparation method of the hyaluronic acid sodium gel injection for the osteoarthrosis chamber according to claim 6 is characterized in that: step 2. in, the mass ratio of cross-linking hyaluronic acid sodium powder and described hyaluronate sodium dry powder is 8: 2 or 6: 4.
8. the preparation method of the hyaluronic acid sodium gel injection for the osteoarthrosis chamber according to claim 7, it is characterized in that: step 3. in to after the gel particle sterilization of collecting, get wherein 45g～50g fill in the disposable syringe of prior sterilization, be settled to 6.0mL with the PBS buffer again and obtain hyaluronic acid sodium gel injection for the osteoarthrosis chamber.
9. the preparation method of the hyaluronic acid sodium gel injection for the osteoarthrosis chamber according to claim 6 is characterized in that: step 1. in the reactant liquor of a the concentration of hyaluronate sodium be 3wt%～4wt%; Step 1. among the c cross-linking hyaluronic acid sodium powder sieving separating adopt 35 orders～45 purpose screen clothes.
10. the gel injection of the preparation method of the hyaluronic acid sodium gel injection for the osteoarthrosis chamber as claimed in claim 6 preparation.
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| CN101538377A (en) * | 2008-03-20 | 2009-09-23 | 上海昊海生物科技有限公司 | Cross-linked hyaluronic acid gel and preparation method thereof |
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Inventor after: He Haoming Inventor after: Jia Chao Inventor after: Zhang Feng Inventor after: Gao Jianjie Inventor before: He Haoming Inventor before: Jia Chao Inventor before: Zhang Feng Inventor before: Zhao Xiaobin |
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Free format text: CORRECT: INVENTOR; FROM: HE HAOMING JIA CHAO ZHANG FENG ZHAO XIAOBIN TO: HE HAOMING JIA CHAO ZHANG FENG GAO JIANJIE |
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