CN102552974A - Gel composition for injection filling of skins and preparation method for gel composition - Google Patents

Abstract

The invention discloses a method for preparing a gel composition for injection filling of skins. The method comprises the following steps of: performing cross-linking reaction on a hyaluronic acid raw material and a cross-linking agent to obtain a gel block, and cleaning and dialyzing the gel block; freezing the dialyzed gel block into a gel ice block, crushing by using a micro-pulverizer, and collecting gel ice particles with different particle size ranges by using a series of mesh sieves with different apertures; and swelling the gel ice particles with different particle size ranges by using a weakly alkaline hyaluronic acid solution respectively so as to close a residual free-state end of the cross-linking agent, and adding an acid solution to obtain the gel composition of which the pH value is within a physiologically acceptable range. By the method, the problem about the residue of the cross-linking agent of which one end is in a bonding state and the other end is still a free-state functional group after cross-linking reaction is solved; and the microgel composition with different particle size levels can be obtained by a simple preparation process so as to be suitable for the injection on different layers of the skins and achieve the beauty effects of filling, removing wrinkles and repairing depressions.

Description

Injection of skin is filled with gel combination and preparation thereof

Technical field

The present invention relates to a kind of gel combination that is suitable for the injection of skin filling and preparation method thereof, especially be applicable to the preparation of the different-grain diameter series gel combination of skin different levels injection.

Background technology

(hyalouronic acid is the linear polymeric polysaccharide that is mutually combined and constituted by glucuronic acid and acetylglucosamine disaccharidase HA) to hyaluronic acid, is the important component that constitutes skin, vitreous body, knuckle synovia and cartilaginous tissue.HA belongs to a kind of physiological material, has characteristics such as high viscoelasticity, good biocompatibility and non-immunogenicity, does not have species specificity, so obtained extensive use in medical treatment, beauty industry.At present, the HA product is mainly as ophthalmology viscoelastic agent, orthopaedics viscoelasticity supplement and post-operation adhesion preventing agent etc., and macromolecule HA solution also can be used as dermal filler, can improve skin appearance or improve lip shape after being expelled to skin inside.But natural HA macromolecule water-solubility is high, and very soon by enzyme and free radical cracking, the half-life in skin histology is merely 12 hours in vivo, has had a strong impact on it and has filled the effect in the beauty treatment at skin histology.And crosslinked HA both can overcome above-mentioned defective, can keep good biocompatibility and the non-immunogenic of HA again, was a kind of comparatively ideal tissue filling material.The crosslinked HA that external at present listing is used for injection of skin filling beauty treatment mainly contains the crosslinked HA gel of divinylsulfone (like Hylaform, Hylaform Plus and Prevelle Silk), the crosslinked HA gel of butanediol diglycidyl ether (BDDE) (like Restylane, Perlane and Juvederm) and the crosslinked HA gel products such as (like Elevess) of carbodiimides.Existing a large amount of patents or patent application both at home and abroad discloses the method for preparing of crosslinked HA; For example US4716154, WO2008034176, WO2008068297 (CN101594891A), US20050281880, US20060194758, WO9704012 (CN1083849C), WO2005085329 (CN100582146C), CN1590444A, CN102188712A, CN102120833A, CN101724164A, CN101153061A, CN101264348A, CN101244290A, CN101502677A etc. disclose the crosslinked HA method of BDDE, and US4582865, US20070036745, WO2005066215 (CN1902232A), WO2006056204 (CN101107270A) and number of patent application 201110393380.6 etc. disclose the crosslinked HA method of DVS.But; In these crosslinked HA methods, the approach of removing residual cross-linker generally is the method that adopts physiological buffer or distilled water to clean, dialyse or with an organic solvent precipitate, wash, and has certain limitation; To an end has been that key knot attitude and the other end still are the cross-linking agent of free state functional group; Can't remove with dialysis or the mode of cleaning, and this kind contains the cross-linking agent of free state functional group and have reactivity, have potential risk after getting in the body.

At present, along with skin being filled deepening continuously of the crosslinked HA research of beauty treatment, scientist and manufacturers have developed the crosslinked HA gel that is fit to be injected in the skin different levels gradually, and their main distinction is that mean particle dia is different.Generally speaking, the microgel particle diameter is big more, and the injection site is darker, injection needle is thick more, is fit to be expelled to subcutaneous or deep skin, corrects darker wrinkle or depression; The microgel particle diameter is more little, and the injection site is shallow more, injection needle is thin more, is fit to be expelled to the skin shallow-layer, corrects light wrinkle or rich lip.For example, the product Restylane and the Perlane of U.S.'s listing, their compositions are identical; Unique difference be mean particle dia, the mean particle dia of Restylane is at the 250-500 mu m range, is fit to inject corium, lower floor; Correct general wrinkle or depression, and the Perlane mean particle dia is bigger, at the 940-1090 mu m range; Be fit to inject corium deep or subcutaneous tissue, correction of severe wrinkle or depression.Method for preparing for crosslinked HA micro gel; Existing multiple patent or patent application relate to; Like CN101502677A, CN101244290A and CN102188712A etc.; But these methods are a kind of preparation of particle diameter rank micro gel, do not relate to a kind of method and prepare multiple particle diameter rank micro gel technology.Related to the technology for preparing multiple particle diameter rank micro gel among the patent application WO2005067994 (CN1893989A); But should steps such as organic solvent deposit, washing have been used in the technology; Increased the risk of product residue organic solvent, and should the preparation process comparatively numerous and diverse.

Summary of the invention

The present invention provides a kind of preparation to be applicable to the method for the gel combination that the injection of skin different levels is filled; Can obtain the serial gel combination of different gel particle size range through the easy manufacture process, and lower the potential risk of the remaining free state of cross-linking agent functional group in the gel.

The method that preparation of the present invention is used in the gel combination of injection of skin filling comprises: (1) hyaluronic acid raw material and cross-linking agent generation cross-linking reaction generate gel piece, and gel piece cleans, dialyses; (2) the back gel piece freezing one-tenth gel ice cube of will dialysing after pulverizing mill is pulverized, is collected the gel ice pellets of different-grain diameter scope through a series of different apertures mesh screen; And the gel ice pellets of (3) different-grain diameter scope respectively in the alkalescence hyaluronic acid solution swelling add acid solution then to obtain the gel combination of pH-value with the remaining free state end of sealing cross-linking agent at the physiology tolerance interval.

In one embodiment, the hyaluronic acid raw material of step (1) is selected from hyaluronate sodium, potassium hyaluronate, calcium hyauronate, hyaluronic acid magnesium, hyaluronic acid ammonium, Curiosin or its mixture; Cross-linking agent is divinylsulfone or the epoxide that is selected from down group: 1; 4-butanediol diglycidyl ether (BDDE), Ethylene glycol diglycidyl ether, 1; 6-hexanediol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether, neopentylglycol diglycidyl ether, 1,2,7; 8-diepoxy octane and 1,3-diepoxy butane; Cross-linking reaction is carried out in the sodium hydroxide solution of 0.05N-1.0N or potassium hydroxide solution, and the concentration of participating in the hyaluronic acid raw material of cross-linking reaction is 4-15%, and cross-linking agent is 1: 10～1: 1 with hyaluronic acid raw materials quality ratio.

Preferably, cross-linking agent is 1, the 4-butanediol diglycidyl ether; The cross-linking reaction temperature is 30-4 ℃, and the response time is 2-12 hour, and the employed liquid of gel that cleans, dialyses is distilled water; The dialysis needed time of gel is 2-7 days, and the number of times of changing dialysis solution is 4-20 time.

In one embodiment, the freezing process of gel piece in the step (2) be gel piece-2 ℃ to-6 ℃ held 12-48 hour, the ambient temperature when gel piece is pulverized is 0-8 ℃.The mesh screen that is respectively 200 μ m, 500 μ m, 1000 μ m and 1500 μ m through average pore size is collected the gel ice pellets, to obtain that particle size range is respectively 0～200 μ m, 200～500 μ m, 500～1000 μ m, 1000～1500 μ m and greater than a series of gel ice pellets of 1500 μ m.

In one embodiment, in step (3), the pH value of alkalescence hyaluronic acid solution is that 8-9, hyaluronic concentration are 1-5%; The mass ratio of alkalescence hyaluronic acid solution and gel ice pellets is 1: 1～1: 4; And gel ice pellets swollen temperature in the alkalescence hyaluronic acid solution is 20-4 ℃, and swelling time is 2-12 hour.

Preferably, in the step (3), the alkalescence hyaluronic acid solution comprises that also concentration is the NaCl of 1.5-4.5%.

Preferably; In the step (3); The acid solution that adds is the anesthetics solution of pH value 2-6; The pH that is the gained gel combination is 6.5 to 7.5, and wherein anesthetics is selected from lignocaine, mepivacaine, prilocaine, bupivacaine, cocaine, procaine, tetracaine or its salt, and the final concentration of anesthetics in gel combination is 0.25-0.3%.

Another aspect of the present invention, the gel combination that is applicable to the injection of skin filling that also provides the preparation in accordance with the present invention of knowing clearly to obtain.

Another aspect of the present invention, the different serial gel combination of gel particle size range that is applicable to skin different levels injection filling that also provides method produced according to the present invention to obtain.

It is key knot state and the other end still is the cross-linking agent residue problem of free state functional group that the inventive method (1) has solved after the cross-linking reaction cross-linking agent one end; (2) use simple preparation technology can obtain other micro gel of different-grain diameter level, be suitable for the injection of skin different levels, the cosmetic result of playing and fill reduce wrinkle, repairing depression.

The specific embodiment

The purpose of this invention is to provide a kind of gel combination method for preparing that injection of skin is filled that is suitable for, reach the serial gel combination that especially is fit to skin different levels filling injection that obtains according to this method with different gel particle diameters.

The present invention's preparation is applicable to that the method for the gel combination that injection of skin is filled comprises step:

(1) hyaluronic acid raw material and cross-linking agent generation cross-linking reaction generate gel piece, and gel piece cleans, dialyses;

(2) the back gel piece freezing one-tenth gel ice cube of will dialysing after pulverizing mill is pulverized, is collected the gel ice pellets of different-grain diameter scope through a series of different apertures mesh screen; And

(3) the gel ice pellets of different-grain diameter scope respectively in the alkalescence hyaluronic acid solution swelling add acid solution then to obtain the gel combination of pH-value with sealing cross-linking agent remaining free state end at the physiology tolerance interval.

The hyaluronic acid raw material is selected from but is not limited to a kind of, two or more the mixture arbitrarily of hyaluronate sodium, potassium hyaluronate, calcium hyauronate, hyaluronic acid magnesium, hyaluronic acid ammonium and Curiosin in the step (1); Preferably clear matter acid sodium, more preferably high-purity, aseptic, low proteic hyaluronate sodium.

Preferably, the initial concentration of hyaluronic acid participation cross-linking reaction is 4-15%.The concentration that this paper relates to " % " is removed other has explanation, all representes w/v.

The cross-linking agent of using always in the medical cross-linked hyaluronic acid gel method for preparing in the prior art, crosslinking process are applicable to the cross-linking reaction among the present invention.In the specific embodiment of the inventive method, cross-linking agent is difunctionality and multifunctional molecule, is selected from epoxide and divinylsulfone; Preferred epoxide is selected from 1,4 butanediol diglycidyl ether, Ethylene glycol diglycidyl ether, 1,6 hexanediol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether, neopentylglycol diglycidyl ether, 1; 2,7,8-diepoxy octane and 1; 3-diepoxy butane, more preferably 1,4 butanediol diglycidyl ether.

Preferably, the addition of cross-linking agent and hyaluronic acid mass ratio are 1: 10-1: 1, be preferably 1: 5-1: 2.

Cross-linking reaction is carried out under alkali condition usually, and for example reaction can be carried out in sodium hydroxide solution that is selected from 0.05N-1.0N or potassium hydroxide solution.Preferably, the cross-linking reaction temperature is a 30-45 ℃ of scope, and the response time is the 2-12h scope.

Gel cleaning of using always in the existing medical cross-linked hyaluronic acid derivatives method for preparing and dialysis process are applicable in the step of the present invention (1).

Preferably, the employed liquid of gel that cleans in the step (1), dialyses is distilled water.Preferably, the time of dialysis gel is 2-7 days, and the number of times of changing dialysis solution is 4-20 time.

The purpose of the freezing process of gel in the step of the present invention (2) is formed with to be beneficial to carries out fine gel ice cube.Preferably, in the specific embodiment, freezing gel process can be to place 12-48 hour at-20 ℃ to-60 ℃ refrigerator-freezers.

Crushing process is meant that the use pulverizing mill is pulverized the solid gel ice cube under low temperature environment.For example, employed pulverizing mill and material contact site are the polishing stainless steel material, and inner corner circular arc is excessive, and the total enclosing operation does not have the material loss, and the refrigeration system minimum temperature of configuration can be to-45 ℃.Preferably, the low temperature environment of pulverizing gel piece is preferably 0-8 ℃ for not being higher than 8 ℃.

Can select the aperture of serial mesh screen according to needed gel particle diameter.In the specific embodiment of the inventive method, use the average pore size of a series of mesh screens to be divided into ranks such as 200 μ m, 500 μ m, 1000 μ m and 1500 μ m in the step (2), come the classification of gel ice pellets.The diameter range of the gel ice pellets that is obtained is respectively 0～200 μ m, 200～500 μ m, 500～1000 μ m, 1000～1500 μ m and greater than 1500 μ m.

Alkalescence hyaluronic acid solution in the step (3) is natural hyaluronic acid solution.The solution pH value is preferably 8-9.Wherein hyaluronic concentration is 1-5%.The optional self-induced transparency matter acid of said natural hyaluronic acid sodium, potassium hyaluronate, calcium hyauronate, hyaluronic acid magnesium, hyaluronic acid ammonium, Curiosin or its mixture, preferably clear matter acid sodium.

Preferably, the mass ratio of natural hyaluronic acid solution of alkalescence and gel ice pellets is 1: 1-1: 4.Preferably, gel ice pellets swollen temperature in the alkalescence hyaluronic acid solution is 20-40 ℃, and swelling time is 2-12 hour.

The inventive method also can be included in adds NaCl to regulate the gel infiltration pressure in the gel combination, the osmotic pressure that makes gel is the physiology tolerance interval.Particularly, can in above-mentioned alkalescence hyaluronic acid solution, add NaCl, concentration is 1.5-4.5%.Preferable osmotic pressure is between 270mOsmol/L to 330mOsmol/L

In the step (3), in the mixture that comprises microgel particle, hyaluronic acid solution, add acid solution and make pH-value reach the physiology tolerance interval, use gel combination thereby finally obtain injection of skin filling of the present invention.

In an embodiment, the neutralize alkalescence of the mixture that comprises microgel particle, hyaluronic acid solution of the acidity of anesthetics solution capable of using itself, making the pH-value of gel combination is the physiology tolerance interval, is preferably pH between 6.5 to 7.5.For example, anesthetics is selected from lignocaine, mepivacaine, prilocaine, bupivacaine, cocaine, procaine, tetracaine, or its esters or solvate, preferably lidocaine hydrochloride.Preferably, the pH value of anesthetics solution is the 2-6 scope.Preferably, behind the adding anesthetics, the final concentration of anesthetics can be 0.25-0.3% in the combined gels solution.

Can obtain serial gel combination according to said method, the hyaluronic acid derivatives in each compositions has different gel particle diameters, thereby should be suitable for skin different levels injection filling by the series gel combination.

Advantage of the present invention is following:

Gel combination in the preparation process, hyaluronic acid (HA) cross-linked gel ice pellets is swollen in the natural HA solution of alkalescence.Under alkaline environment, the cross-linking agent free state end in the gel can be further and the hydroxyl generation cross-linking reaction of natural HA, forms ehter bond; In addition, pulverize that back gel particle diameter is less, infiltration is forced down, help natural HA and be penetrated into micro gel inside and carry out cross-linking reaction.The result of cross-linking reaction reduced the free state terminal number amount of cross-linking agent, thereby reduced the potential risk after cross-linking agent is implanted in the body.

Simple technologies such as that the preparation process of crosslinked HA microgel particle adopts is freezing, pulverizing and sieving; Easily obtained other micro gel of different-grain diameter level; Wherein small particle diameter rank gel is fit to be expelled to the dermis of skin shallow-layer; Middle particle diameter rank gel is fit to be expelled to the dermis of skin middle level to deep layer, and big particle diameter rank gel is fit to be expelled to subcutaneous, the cosmetic result of all playing and fill reduce wrinkle, repairing depression.Therefore, the present invention makes easily and is suitable for the serial gel combination that the injection of skin different levels is filled.

Owing to used the natural HA solution of alkalescence to mix with the gel ice pellets, include a small amount of natural HA in the gel combination that the present invention makes, inject resistance in the time of can reducing injection product.In addition; Because the small-amount free HA that contains in the gel combination; And crosslinked HA molecule constantly is degraded into free HA by body in injection back several weeks or several months, has the effect of inducing the fibroblast generation and suppressing extracellular matrix degradation, can improve the filling cosmetic result of injection site.

Include a small amount of anesthetics in the gel combination of the present invention, but the pain of injection site, reduction of patient injection back has improved the comfortableness that product uses.

Method of testing

1. microgel particle measuring diameter

To the crosslinked HA micro gel for preparing, use laser light scattering instrument (LS 300) to measure its particle size and distribution thereof.

2. the mensuration of content of crosslinking agent

Take by weighing an amount of gel combination or crosslinked HA gel; In the physiological balance liquid that contains hyaluronidase 1000U/ml; 3 ℃ of calorstats are placed to cross-linked hyaluronic acid gel degrade fully after, use gas chromatography to detect the content of crosslinking agent in the degradation solution, extrapolate content of crosslinking agent in the gel then.

3. cell toxicity test

Method according to GB/T16886.5-2003 (lixiviating solution method) regulation is carried out cell toxicity test.

4. Implantation Test

Cavia porcellus intradermal injection study model is adopted in this test, and sample and control material are carried out intradermal injection, and in the 2nd, 4,8,12 time-of-week nodes are measured the height and the diameter in each injection site (each 6 sites of each sample), have or not the edema situation with detection; Simultaneously, at per observing time of node, put to death experimental animal and also do Histological assessment, to detect in the tissue whether have inflammatory reaction.

Injecting the last hour hair with each animal left side and right side cuts off.6 injections of every animals received, every side 3 points.Test material and control material randomize all in each animal.Each injection site is all accepted the dose ejection of 0.2ml.Distance is about 2cm between each injection site.

Embodiment

Further specify the present invention through embodiment below, but be not limited to following instance.

Material

The used hyaluronic acid raw material of following example all derives from bacterial fermentation.

Embodiment 1

5.0g hyaluronic acid (HA) raw material (molecular weight is 1,000,000 dalton) is dissolved in the 0.2N NaOH solution of 40ml, the dissolving back adds 1,4 butanediol diglycidyl ether 3ml, fully stirs, and places 37 ℃ of calorstats 4 hours, promptly forms crosslinked HA gel piece.With distilled water cleaning, dialysis gel piece, in subsequently 3 days, change 6 times dialysis solution.Remove distilled water; Crosslinked HA gel piece is put freezing 24h in-50 ℃ of refrigerator-freezers, take out crosslinked HA ice cube, in 4 ℃ of environment; The pulverizing mill temperature is set to-20 ℃; Using pulverizing mill to pulverize ice cube fast, is the rustless steel mesh screen of 200 μ m, 500 μ m, 1000 μ m and 1500 μ m through average pore size successively then, and the different gel ice pellets of difference collection cut size rank.Respectively with the amount of the crosslinked HA ice pellets of per 100 grams add that pH is 8.5, HA concentration is 4%, NaCl concentration is 1.5% solution 50ml, stir back 30 ℃ and place 6h.Add pH and be 5.5, lidocaine hydrochloride concentration is 1.2%, NaCl concentration is 1.5% solution 50ml.Jolting 24h in four-dimensional bottle swingging machine makes the gel combination sample that obtains present embodiment after the abundant balance of each component.

Embodiment 2

5.0gHA raw material (molecular weight is 1,000,000 dalton) is dissolved in the 0.2N NaOH solution of 50ml, the dissolving back adds 1,4 butanediol diglycidyl ether 4ml, fully stirs, and places 32 ℃ of calorstats 6 hours, promptly forms crosslinked HA gel piece.With distilled water cleaning, dialysis gel piece, in subsequently 3 days, change 9 times dialysis solution.Remove distilled water; Crosslinked HA gel piece is put freezing 48h in-50 ℃ of refrigerator-freezers, take out crosslinked HA ice cube, in 4 ℃ of environment; The pulverizing mill temperature is set to-15 ℃; Using pulverizing mill to pulverize ice cube fast, is the rustless steel mesh screen of 200 μ m, 500 μ m, 1000 μ m and 1500 μ m through average pore size successively then, and the different gel ice pellets of difference collection cut size rank.With the amount of the crosslinked HA ice pellets of per 100 grams add that pH is 8.9, HA concentration is 3.5%, NaCl concentration is 1.5% solution 50ml, stir back 35 ℃ and place 6h.Add pH and be 5.4, lidocaine hydrochloride concentration is 1.2%, NaCl concentration is 1.5% solution 50ml.Jolting 24h in four-dimensional bottle swingging machine makes the gel combination sample that obtains present embodiment after the abundant balance of each component.

Comparative Examples 1 adopts existing method directly to squeeze broken to crosslinked HA gel

5.0gHA raw material (molecular weight is 1,000,000 dalton) is dissolved in the 0.2N NaOH solution of 40ml, the dissolving back adds 1,4 butanediol diglycidyl ether 3ml, fully stirs, and places 37 ℃ of calorstats 4 hours, promptly forms crosslinked HA gel piece.With distilled water cleaning, dialysis gel piece, in subsequently 3 days, change 6 times dialysis solution.Remove distilled water, using and squeezing crushing device is that the stainless steel mesh of 500 μ m squeezes the broken micro gel that is to crosslinked HA gel piece through the aperture.With the amount of the crosslinked HA micro gel of per 100 grams add that pH is 8.5, HA concentration is 4%, NaCl concentration is 1.5% solution 50ml, stir back 30 ℃ and place 6h.Add pH and be 5.5, lidocaine hydrochloride concentration is 1.2%, NaCl concentration is 1.5% solution 50ml.Jolting 24h in four-dimensional bottle swingging machine makes the gel combination sample that obtains this Comparative Examples after the abundant balance of each component.

Use laser light scattering instrument (LS 300) to measure the gel combination sample of embodiment 1, embodiment 2, Comparative Examples 1, reach microgel particle size and distribution thereof among commercially available prod Restylane (auspicious indigo plant) and the Perlane, the result sees table 1.

Table 1

Visible by table 1, the inventive method can prepare a series of other gel combinations of different-grain diameter level that have, and the injection that can be suitable for the skin different levels is filled.And Comparative Examples 1 adopts traditional single aperture to squeeze broken method, only prepares other sample of a kind of grain-size grade.

Comparative Examples 2 gel ice pellets are directly with the NaCl solution swelling that contains natural HA, anesthetics

5.0gHA raw material (molecular weight is 1,000,000 dalton) is dissolved in the 0.2N NaOH solution of 40ml, the dissolving back adds 1,4 butanediol diglycidyl ether 3ml, fully stirs, and places 37 ℃ of calorstats 4 hours, promptly forms crosslinked HA gel piece.With distilled water cleaning, dialysis gel piece, in subsequently 3 days, change 6 times dialysis solution.Remove distilled water; Crosslinked HA gel piece is put freezing 24h in-50 ℃ of refrigerator-freezers, take out crosslinked HA ice cube, in 4 ℃ of environment; The pulverizing mill temperature is set to-20 ℃; Using pulverizing mill to pulverize ice cube fast, is the rustless steel mesh screen of 200 μ m, 500 μ m, 1000 μ m and 1500 μ m through average pore size successively then, and the different gel ice pellets of difference collection cut size rank.Respectively with the amount of the crosslinked HA ice pellets of per 100 grams add that pH is 7.5, HA concentration is 2%, lidocaine hydrochloride concentration is 0.6%, NaCl concentration is 1.5% solution 100ml; Jolting 48h in four-dimensional bottle swingging machine; After making the abundant balance of each component, obtain this Comparative Examples gel combination sample.

Comparative Examples 3 gel ice pellets are directly with the NaCl solution swelling that contains natural HA, anesthetics

5.0gHA raw material (molecular weight is 1,000,000 dalton) is dissolved in the 0.2N NaOH solution of 50ml, the dissolving back adds 1,4 butanediol diglycidyl ether 4ml, fully stirs, and places 32 ℃ of calorstats 6 hours, promptly forms crosslinked HA gel piece.With distilled water cleaning, dialysis gel piece, in subsequently 3 days, change 9 times dialysis solution.Remove distilled water; Crosslinked HA gel piece is put freezing 24h in-50 ℃ of refrigerator-freezers, take out crosslinked HA ice cube, in 4 ℃ of environment; The pulverizing mill temperature is set to-15 ℃; Using pulverizing mill to pulverize ice cube fast, is the rustless steel mesh screen of 200 μ m, 500 μ m, 1000 μ m and 1500 μ m through average pore size successively then, and the different gel ice pellets of difference collection cut size rank.Respectively with the amount of the crosslinked HA ice pellets of per 100 grams add that pH is 7.3, HA concentration is 2%, lidocaine hydrochloride concentration is 0.6%, NaCl concentration is 1.5% solution 100ml; Jolting 48h in four-dimensional bottle swingging machine; After making the abundant balance of each component, obtain the gel combination sample of this Comparative Examples.

Take off the row test specimen: the gel combination that 1. makes through 500 μ m screen clothes among the embodiment 1,2. among the embodiment 2 through gel combination that 500 μ m screen clothes make, 3. (be the natural HA product of selling on the market through gel combination that 500 μ m screen clothes make, the gel combination and the 5. natural HA that 4. make through 500 μ m screen clothes in the Comparative Examples 3 in the Comparative Examples 2; Commodity are called Shi Peike; Produce by Shandong Freda Biopharm Co., Ltd.), carry out cell toxicity test and Implantation Test.Wherein, 1., 2., 3. and 4. (121 ℃ 30min), 5. is the control material group to sample through steam high-voltage sterilizing before making an experiment.Testing result is seen table 2.

Table 2

Table 2 shows that in embodiment of the invention sample and the comparative example, the content of crosslinking agent B DDE is all less than 1 μ g/g, and according to related data, BDDE concentration is lower than 2 μ g/g among the crosslinked HA, and non-carcinogenesis, hereditary-less toxicity belong to safety range.

Cell toxicity test result shows that the cytotoxicity of all test specimens all is not more than 1 grade, all meets the regulation of national sector standard to bio-medical material, can be used in the organism.

The Implantation Test result shows that the edema situation of embodiment of the invention sample and tissue reaction and control material group do not have significant difference, and the inflammatory reaction in the comparative example overweights the control material group.Cause the reason of above-mentioned result of the test; It possibly be the content of crosslinking agent seldom (qualified) of dissociating in the comparative example because content of crosslinking agent mensuration and cell toxicity test result show; But also having cross-linking agent one end is key knot state and the other end still is free state functional group; The free state end of cross-linking agent can react with body after implanting, and causes inflammation to produce.And the free state end owing to cross-linking agent is closed in the embodiment of the invention, so inflammatory reaction is with the matched group zero difference.

Claims (10)

1. a method for preparing the gel combination that is applicable to that injection of skin is filled the steps include:

(1) hyaluronic acid raw material and cross-linking agent generation cross-linking reaction generate gel piece, and gel piece cleans, dialyses;

(2) the back gel piece freezing one-tenth gel ice cube of will dialysing after pulverizing mill is pulverized, is collected the gel ice pellets of different-grain diameter scope through a series of different apertures mesh screen; And

(3) the gel ice pellets of different-grain diameter scope respectively with alkalescence hyaluronic acid solution swelling with sealing cross-linking agent remaining free state end, add acid solution then to obtain the gel combination of pH-value at the physiology tolerance interval.

2. the method for claim 1, wherein in step (1), the hyaluronic acid raw material is selected from hyaluronate sodium, potassium hyaluronate, calcium hyauronate, hyaluronic acid magnesium, hyaluronic acid ammonium, Curiosin or its mixture; Cross-linking agent is divinylsulfone or the epoxide that is selected from down group: 1; 4-butanediol diglycidyl ether, Ethylene glycol diglycidyl ether, 1; 6-hexanediol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether, neopentylglycol diglycidyl ether, 1,2,7; 8-diepoxy octane and 1,3-diepoxy butane; Cross-linking reaction is carried out in the sodium hydroxide solution of 0.05N-1.0N or potassium hydroxide solution, and the concentration of participating in the hyaluronic acid raw material of cross-linking reaction is 4-15%, and cross-linking agent is 1: 10～1: 1 with hyaluronic acid raw materials quality ratio.

3. method as claimed in claim 2, wherein, cross-linking agent is 1; The 4-butanediol diglycidyl ether; The cross-linking reaction temperature is 30-4 ℃, and the response time is 2-12 hour, and the employed liquid of gel that cleans, dialyses is distilled water; The dialysis needed time of gel is 2-7 days, and the number of times of changing dialysis solution is 4-20 time.

4. the method for claim 1, wherein in step (2), the freezing process of gel piece be gel piece-2 ℃ to-6 ℃ held 12-48 hour, the ambient temperature when gel piece is pulverized is 0-8 ℃.

5. the method for claim 1; Wherein in step (2); The a series of mesh screens that are respectively 200 μ m, 500 μ m, 1000 μ m and 1500 μ m through average pore size are collected the gel ice pellets, to obtain that the gel particle size range is respectively 0～200 μ m, 200～500 μ m, 500～1000 μ m, 1000～1500 μ m and greater than a series of gel ice pellets of 1500 μ m.

6. the method for claim 1, wherein in step (3), the pH value of alkalescence hyaluronic acid solution is that 8-9, hyaluronic concentration are 1-5%; The mass ratio of alkalescence hyaluronic acid solution and gel ice pellets is 1: 1～1: 4; And gel ice pellets swollen temperature in the alkalescence hyaluronic acid solution is 20-4 ℃, and swelling time is 2-12 hour.

7. method as claimed in claim 6, alkalescence hyaluronic acid solution comprise that also concentration is the NaCl of 1.5-4.5%.

8. the method for claim 1; Wherein in step (3); The acid solution that adds is the anesthetics solution of pH value 2-6; The pH that is the gained gel combination is 6.5 to 7.5, and wherein anesthetics is selected from lignocaine, mepivacaine, prilocaine, bupivacaine, cocaine, procaine, tetracaine or its salt, and the final concentration of anesthetics in gel combination is 0.25-0.3%.

9. be applicable to the gel combination that injection of skin is filled, make through each method of claim 1-8.

10. be applicable to the different serial gel combination of gel particle size range that skin different levels injections is filled, make through each method of claim 1-8.