CN102836465A - Silk-fibroi and hyaluronic-acid (HA) composite gel for injection and preparation and application thereof - Google Patents
Silk-fibroi and hyaluronic-acid (HA) composite gel for injection and preparation and application thereof Download PDFInfo
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Abstract
The invention discloses a silk-fibroi and hyaluronic-acid (HA) composite gel for injection and the preparation and application thereof. The composite gel is prepared from the following raw materials by weight: 1 part of silk fibroi microspheres, 1-50 parts of hyaluronate and 0.08-4 parts of crosslinking agent. Compared with the traditional hyaluronic-acid purely-crosslinked gel, due to the addition of the silk fibroi particles, the degradation rate of the composite gel is reduced effectively, the tissue repairing and filling time is prolonged and the effectiveness of the materials is improved, and due to the addition of the HA, the water absorption, the lubrication degree and the water storage function of the materials are improved. Compared with the silk fibroi particles and the HA particles mixed by adopting the pure physical method, because the silk fibroi particles are wrapped in the gel HA particles, the silk fibroi particles and the HA particles are mixed more uniformly, thus the prepared gel particles have low inflammatory response and good biocompatibility, and the safety of the materials is improved.
Description
(1) technical field
The present invention relates to the Medical Cosmetic Collagen Injection packing material, particularly a kind of injection fibroin albumen hyaluronic acid pluralgel and method for preparing and the application in soft tissue filling material.
(2) background technology
In tissue repairing, tissue enhancement, the growth of non-operative treatment is rapid at present, and proportion has surpassed 80%; Because local injection belongs to the minimally-invasive treatment method, only need the partial relaxation degree is improved in some drugs and the lax soft tissue part of chemicals injecting structure, improve the ability of local tolerance pressure, thereby make its normal function of this organized renewing or outward appearance.
Along with the continuous development of science and technology, the tissue filling material is also more and more, can they be divided into biomaterial class and injection cell product class according to its composition and biological property.
Adopting injection cell is the Therapeutic Method that has application prospect, and cell can synthesize the justacrine extracellular matrix, can promote the synthetic of collagen molecules, recovers defective tissue.But because mostly cell products is liquid, be difficult to control its distribution after being expelled to local organization, can not plastotype, influence tissue repairing, enhanced effect.
The biomaterial of having reported that is used for tissue repairing, enhancing class is existing a lot, and wherein hyaluronic acid is comparatively outstanding.Hyaluronic acid is the straight chain shape macromolecule polysaccharide that β-D-N acetylglucosamine and β-D-glucuronic acid mutually combine, owing to do not have kind and internal organs specificity.Hyaluronic acid has special hydrodynamic performance, and this makes it have height viscoelasticity, plasticity, permeability and excellent biological compatibility.It also has the tissue adhesion of preventing, significantly reduces postoperative complication, simultaneously, the promotion wound healing is arranged, and reduces the advantage of cicatrix.After in being implanted to human body, do not cause allosome rejection and anaphylaxis.
Natural ectogenic hyaluronan molecule amount is greatly about 100,000 ~ 5,000,000 Dalton, RT in vivo greatly about about 3 ~ 15 days, largely limit its application in some field.
Patent CN1590444A adopts hyaluronic acid (HA) and cross-linking agent (BDDE) to react 2-24 hour with proper proportion, forms hyaluronic acid derivatives.Single hyaluronic acid derivatives will not reach the purpose of long-term filling because of the metabolism decomposition and absorption after a while as soft tissue filling material.
The method that patent CN101502676A adopts is that polymethyl methacrylate (PMMA) microsphere and cross-linked hyaluronic acid gel microgranule are mixed.Because the existence of PMMA microsphere makes product retaining in when injected organism tissue have persistency.Yet because PMMA is a kind of high molecular polymer, can produce foreign body to local organization stimulates, and biocompatibility is relatively poor relatively.
Research shows; Silkworm silk is made up of about 75% fibroin and about 25% sericin, and it is the native protein that silkworm comes out in the silk glands endocrine, is made up of 18 seed amino acids; Mainly form, have certain similar with human collagen albumen by multiple Gly-Ala-Gly-Ala-Ser polyembryony unit.Its avirulence, nonirritant are that a kind of degraded is slow, and metastable biomaterial, are widely used in fields such as biomaterial subject, organizational project and drug release.
There is researcher to adopt methods such as supersonic induced or polymer electrostatic interaction to prepare fibroin albumen-hyaluronic acid pluralgel.Compare with simple hyaluronic acid derivatives, prolonged the degradation time of gel greatly.The right pluralgel that adopts method for preparing only depends on simple fibroin albumen, or the physical action between fibroin albumen and the hyaluronic acid, and hyaluronic acid contents is less, and the water absorption of pluralgel is relatively poor.
Therefore, seek a kind of method for preparing fibroin albumen and hyaluronic acid pluralgel, can make material tool good biocompatibility, and have good water absorption, degradation speed slowly concurrently.So seek a kind of long fibroin albumen and hyaluronic acid composite of retention time in good biocompatibility, water absorption, the body that have concurrently, tissue repairing that can be permanently effective, reinforcing material become starting point of the present invention.
(3) summary of the invention
The present invention seeks to overcome the deficiency of above-mentioned prior art; Adopt the method for preparing hyaluronic acid, fibroin albumen composite material; A kind of good biocompatibility, mechanical property and good water absorption, tissue repairing that can be permanently effective, reinforcing material of having concurrently is provided.
The technical scheme that the present invention adopts is:
A kind of injection fibroin albumen hyaluronic acid pluralgel, said pluralgel is made up of following quality proportion raw material: 1 part of fibroin albumen microsphere, 0.08 ~ 4 part of 1 ~ 50 part of hyaluronate and cross-linking agent; Said fibroin albumen microsphere be silk fibroin protein solution with mass concentration 5% after leaving standstill gel under 25 ℃, homogenizing 5min sieves under the 24000rpm/min condition, collects the fibroin albumen microsphere of 200 ~ 325 mesh sieves; Said cross-linking agent is divinylsulfone or 2-glycidyl ether compound; Be preferably the mixing of one or more arbitrary proportions in divinylsulfone, Ethylene glycol diglycidyl ether, butanediol diglycidyl ether or the polypropylene glycol diglycidyl ether, most preferably butanediol diglycidyl ether.
Said hyaluronate is preferably the mixing of one or more arbitrary proportions in hyaluronate sodium, potassium hyaluronate, the hyaluronic acid magnesium, more preferably hyaluronate sodium.
Further, said pluralgel is made up of following quality proportion raw material: 1 part of fibroin albumen microsphere, 0.8 ~ 2.4 part of 10 ~ 30 parts of hyaluronates and cross-linking agent.
Further, said pluralgel is made up of following quality proportion raw material: 1 part of fibroin albumen microsphere, 2.4 parts of 30 parts of hyaluronates and cross-linking agent.
Further, said pluralgel is made up of following quality proportion raw material: 1 part of fibroin albumen microsphere, 1.6 parts of 20 parts of hyaluronates and cross-linking agent.
Further, said pluralgel is made up of following quality proportion raw material: 1 part of fibroin albumen microsphere, 0.8 part of 10 parts of hyaluronates and cross-linking agent.
Further, the method for preparing of injection fibroin albumen hyaluronic acid pluralgel according to the invention is: the preparation of (1) fibroin albumen microsphere: Bombyxmori Linnaeus silkworm silk is joined in the aqueous sodium carbonate of mass concentration 0.2% 96 ~ 100 ℃ of water-bath 60min; Filter; Get filter cake and repeat water-bath operation 3 times, get that the lithium bromide water solution with 9mol/L dissolves behind the last filtering filtration cakes torrefaction, at 60 ℃ of water-bath 6h; Centrifugal 5min under the 8000rpm condition then; Collecting supernatant, is that 8000 ~ 14000 bag filter is at room temperature dialysed with molecular cut off (MW), is dialysis solution with the purified water; Magnetic agitation dialysis 3 days; Get the silk fibroin protein solution (get a certain amount of described trapped fluid dry 4h in 80 ℃ of baking ovens and calculate the water content of trapped fluid, and then the trapped fluid water is mixed with the silk fibroin protein solution of mass concentration 5%) that the trapped fluid water is mixed with mass concentration 5% to constant weight; After leaving standstill gel under 25 ℃, homogenizing 5min sieves under the 24000rpm/min condition, collects the fibroin albumen microgranule of 200 ~ 325 mesh sieves with the silk fibroin protein solution of mass concentration 5%; The volumetric usage of said aqueous sodium carbonate is counted 67.5 ~ 125ml/g (preferred 67.5ml/g) with the Bombyxmori Linnaeus silk quality, and the volumetric usage of said lithium bromide water solution is counted 5 ~ 6.25ml/g with the Bombyxmori Linnaeus silk quality; (2) preparation of injection fibroin albumen hyaluronic acid pluralgel: the hyaluronic acid saline solution that the hyaluronate of formula ratio is mixed with 0.1g/ml with the sodium hydrate aqueous solution of mass concentration 1%; Fibroin albumen microgranule with formula ratio joins abundant mixing in the above-mentioned hyaluronic acid saline solution then; Add formula ratio cross-linking agent mixing again; Leave standstill 2 ~ 8h under 25 ~ 60 ℃ of conditions; Form fibroin albumen hyaluronic acid composite gel material; Fibroin albumen hyaluronic acid composite gel material is placed the bag filter of molecular cut off (MW) 8000 ~ 14000, is that 7.4 PBS buffer is that dialysis solution carries out dialysis treatment with pH, gets composite gel material (the being trapped fluid) homogenizing 5 ~ 15min under the 24000rpm/min condition after the dialysis treatment; Cross 60 mesh sieves, collection cut size is that the granule of 60 mesh sieves is said injection fibroin albumen hyaluronic acid pluralgel; Said hyaluronate is the mixing of one or more arbitrary proportions in hyaluronate sodium, potassium hyaluronate, the hyaluronic acid magnesium; Preferably clear matter acid sodium; Said cross-linking agent is the mixing of one or more arbitrary proportions in divinylsulfone, Ethylene glycol diglycidyl ether, butanediol diglycidyl ether or the polypropylene glycol diglycidyl ether, preferred butanediol diglycidyl ether.
Further, step (2) preferably leaves standstill under 30 ~ 40 ℃ of conditions after adding the cross-linking agent mixing.
Further, step (2) preferably leaves standstill under 4 ~ 6h condition after adding the cross-linking agent mixing.
Advantages such as the present invention provides the application of said injection fibroin albumen hyaluronic acid pluralgel in soft tissue filling material, and it has uniform particle diameter, is easy to inject, compares with simple HA injected gel, and RT is long.
Said soft tissue filling material is meant little shaping packing materials such as face crinkle-removing.
The average particle size range of fibroin albumen microsphere is 1 ~ 120 μ m, preferred 20 ~ 100 μ m, and more preferably 50 ~ 75 μ m, because the fibroin albumen microsphere be white in solution, opaque, therefore, pluralgel outward appearance of the present invention presents milky.
Compared with prior art, fibroin albumen hyaluronic acid pluralgel of the present invention has following remarkable advantage:
1) the present invention and the simple crosslinked gel phase ratio of conventional transparent matter acid, the external resistance to enzymolysis ability of fibroin albumen hyaluronic acid pluralgel significantly improves, external resistance to enzymolysis evidence; At same enzyme liquid, under the identical time effect, the hyaluronic external resistance to enzymolysis ability in the fibroin albumen hyaluronic acid pluralgel increases along with the particulate increase of fibroin albumen; Can find thus; The particulate adding of fibroin albumen has effectively slowed down the degradation speed of pluralgel, prolongs it in tissue repairing; The action time of filling, improved the effectiveness of material;
2) the present invention compares with simple fibroin albumen granular materials, and the adding of HA has increased the water absorption of material, improves the lubricity and the water storage function of material, can significantly increase the filling effect of material;
3) the present invention compares with the HA granule with simple physical method silk union fibroin granule; Because the fibroin albumen granule is wrapped in the HA gel particle, both mixing are more even, and the in vivo test of SD rat proves; Fibroin albumen hyaluronic acid pluralgel is the same with simple cross-linking hyaluronic acid gel; Inflammatory reaction is little, good biocompatibility, and this has improved the safety of material.
(4) description of drawings
Fig. 1 glucuronic acid standard curve;
The external anti-degradation capability of Fig. 2 fibroin albumen hyaluronic acid pluralgel: sample 1 is the fibroin albumen hyaluronic acid pluralgel of embodiment 1 preparation; Sample 2 is the fibroin albumen hyaluronic acid pluralgel of embodiment 2 preparations, and sample 3 is the fibroin albumen hyaluronic acid pluralgel of embodiment 3 preparations;
Fig. 3 fibroin albumen hyaluronic acid pluralgel is implanted the tissue slice microgram after two weeks in vivo; The fibroin albumen hyaluronic acid pluralgel that A prepares for embodiment 1 is implanted the tissue slice microgram after two weeks in vivo, and the fibroin albumen hyaluronic acid pluralgel that B prepares for embodiment 1 is implanted the tissue slice microgram after eight weeks in vivo;
Fig. 4 fibroin albumen hyaluronic acid pluralgel and hyaluronic acid derivatives are implanted the tissue slice microgram after two weeks in vivo; A ~ D is the HE dyeing picture that embodiment 1 ~ 4 preparation fibroin albumen hyaluronic acid pluralgel and hyaluronic acid derivatives body were implanted into for two weeks, and E is the HE dyeing picture after check sample (PBS) body was implanted into for two weeks.
(5) specific embodiment
Below in conjunction with specific embodiment the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1: fibroin albumen hyaluronic acid pluralgel
(1) preparation of fibroin albumen microsphere: 40g Bombyxmori Linnaeus silkworm silk joins in the aqueous sodium carbonate of 2700ml mass concentration 0.2%, and 96 ~ 100 ℃ of water-bath 60min filter; Get filter cake and repeat water-bath 3 times, get last filtering filter cake lithium bromide water solution 200ml with 9mol/L after 60 ℃ of oven dry and slowly dissolve, at 60 ℃ of water-bath 6h; The centrifugal 5min of 8000rpm gets supernatant then, with the bag filter (MW8 that packs into of the supernatant after the centrifugal treating; 000 ~ 14,000), with the purified water is dialysis solution; Trapped fluid is got in magnetic agitation dialysis three days, and to use purified water to be mixed with mass concentration be 5% silk fibroin protein solution.
Under 25 ℃ of conditions, above-mentioned 5% silk fibroin protein solution left standstill forms white gels material 5g, with gel rubber material with homogenizer (T25; IKA) behind homogenizing 5min under the 24000rpm/min condition; Obtain the different fibroin albumen gel particle 5g of diameter, filter, collected the fibroin albumen granule 3.5g of 200 mesh sieves; Fibroin albumen particle drying to the constant weight that takes by weighing small collected is confirmed the particulate water content of fibroin albumen, and definite fibroin albumen granular mass concentration is 3.23%.
(2) fibroin albumen hyaluronic acid pluralgel: the 0.7g hyaluronate sodium is dissolved in 7ml mass concentration 1% sodium hydroxide solution processes the 0.1g/ml sodium hyaluronate solution; And in sodium hyaluronate solution, add the fibroin albumen granule 0.712g (0.023/0.0323=0.712g that step (1) obtains; Fibroin albumen granule dry weight is 0.023g); Fully mix the BDDE (1, the 4-butanediol diglycidyl ether) that the back adds 56 μ l (0.056g), behind the mix homogeneously; Place to leave standstill under 40 ℃ of conditions and carried out crosslinkedly in 4 hours, can form fibroin albumen-HA composite gel material.
Above-mentioned fibroin albumen-HA composite gel material is joined MW8, and 000-14 is in 000 the bag filter; With pH value is that 7.4 PBS buffer is a dialysis solution, under magnetic agitation, carries out dialysis treatment, with the composite gel material after the dialysis treatment with homogenizer behind homogenizing 10min under the 24000rpm/min condition; Place to push in the syringe to make gel pass through 60 mesh sieves, collect composite gel particle, behind 120 ℃ of high temperature and high pressure steams sterilization 15min; Carry out aseptic subpackaged, in the disposable syringe of packing into.Promptly obtain the fibroin albumen hyaluronic acid pluralgel 35g of injection, can be used as hypodermic products.Get fibroin albumen hyaluronic acid pluralgel with mass ratioes such as distilled water work dilution (proportioning is seen shown in the table 1), behind the mix homogeneously, room temperature (25 ℃) leaves standstill half an hour; Measure the pH value of solution; Each sample triplicate, and average as the pH value of sample, the result sees shown in the table 1.
Embodiment 2 fibroin albumen hyaluronic acid pluralgel
(1) according to the described method of embodiment 1 step (1), prepared the fibroin albumen granule 3.5g of 200 mesh sieves, mass concentration is 3.23%.
(2) the 0.7g hyaluronate sodium is dissolved in 7ml mass concentration 1% sodium hydroxide solution processes the 0.1g/ml sodium hyaluronate solution; And in hyaluronic acid solution, add the fibroin albumen granule 1.0836g (0.035/0.0323=1.0836g) that step (1) obtains; Fully mix the BDDE that the back adds 56 μ l; Behind the mix homogeneously, place to leave standstill under 40 ℃ of conditions and carried out crosslinkedly in 6 hours, form fibroin albumen hyaluronic acid composite gel material.
Above-mentioned fibroin albumen hyaluronic acid composite gel material is carried out dialysis treatment, and other is operated with embodiment 1, obtains the fibroin albumen hyaluronic acid pluralgel 35g of injection, can be used as the tissue filling injection product.Get fibroin albumen hyaluronic acid pluralgel with mass ratioes such as distilled water work dilution (proportioning is seen shown in the table 1), behind the mix homogeneously, room temperature (25 ℃) leaves standstill half an hour; Measure the pH value of solution; Each sample triplicate, and average as the pH value of sample, the result sees shown in the table 1.
Embodiment 3: fibroin albumen hyaluronic acid pluralgel
(1) according to the described method of embodiment 1 step (1), prepared the fibroin albumen granule 3.5g of 200 mesh sieves, mass concentration is 3.23%.
(2) the 0.7g hyaluronate sodium is dissolved in the sodium hyaluronate solution of processing 0.1g/ml in 7ml mass concentration 1% sodium hydroxide solution; And in sodium hyaluronate solution, add 2.167g (0.07/0.0323=2.167g) fibroin albumen granule; Fully mix the BDDE that the back adds 56 μ l; Behind the mix homogeneously, place to leave standstill under 40 ℃ of conditions and carried out crosslinkedly in 5 hours, form fibroin albumen hyaluronic acid composite gel material.
Fibroin albumen hyaluronic acid composite gel material is carried out dialysis treatment, and other is operated with embodiment 1, obtains the fibroin albumen hyaluronic acid pluralgel 35g of injection, can be used as subcutaneous filling injection product.Get fibroin albumen hyaluronic acid pluralgel with mass ratioes such as distilled water work dilution (proportioning is seen shown in the table 1), behind the mix homogeneously, room temperature (25 ℃) leaves standstill half an hour; Measure the pH value of solution; Each sample triplicate, and average as the pH value of sample, the result sees shown in the table 1.
Embodiment 4: the preparation of hyaluronic acid derivatives
The 0.7g hyaluronate sodium is dissolved in the sodium hyaluronate solution of processing 0.1g/ml in 7ml mass concentration 1% sodium hydroxide solution; Fully mix the back and add 56 μ l BDDE; Behind the mix homogeneously, place to leave standstill under 40 ℃ of conditions and carried out crosslinkedly in 5 hours, can obtain hyaluronic acid derivatives.
After hyaluronic acid derivatives carried out dialysis treatment, other was operated with embodiment 1, promptly obtains the hyaluronic acid derivatives 35g of injection.Get fibroin albumen hyaluronic acid pluralgel with mass ratioes such as distilled water work dilution (proportioning is seen shown in the table 1), behind the mix homogeneously, room temperature (25 ℃) leaves standstill half an hour; Measure the pH value of solution; Each sample triplicate, and average as the pH value of sample, the result sees shown in the table 1.
Table 1 fibroin albumen hyaluronic acid pluralgel pH value
Table 1 can find out, the pH value of the prepared fibroin albumen hyaluronic acid pluralgel of embodiment 1 ~ 4, hyaluronic acid derivatives is all 7.4.
Embodiment 5: external anti-degradation capability test
Cross-linked-hyaluronic acid can resolve into glucuronic acid through behind the enzymolysis, and glucuronic acid and the effect of carbazole reagent produce reddish violet, and the depth of color is directly proportional with the content of glucuronic acid.Can confirm the content of glucuronic acid through light absorption value.
Through investigating in the fibroin albumen hyaluronic acid pluralgel that different proportion fibroin albumen addition processes; What of glucuronic acid content; Judge the external resistance to enzymolysis ability of pluralgel, thereby judge the external resistance to enzymolysis ability of pluralgel and simple cross-linking hyaluronic acid gel.
Specific embodiments is following:
Carry out the fibroin albumen hyaluronic acid pluralgel of embodiment 1 ~ 3 preparation the mensuration of external anti-degradation capability.
(1) drafting of standard curve
The glucal standard acid solution of water preparation 70,60,50,40,30 μ g/ml is got the 0.5ml titer and is placed test tube, with the 0.5ml distilled water as contrast.Standard pipe and control tube are placed ice-water bath (0 ℃) together; In every pipe, add sodium tetraborate sulphuric acid 2.5ml lentamente respectively with glass pipet; The limit edged shakes up (note, need abundant mixing), takes out after placing boiling water bath to boil 20min; Be cooled to room temperature (can ice bath, quicken cooling).Add 0.1% carbazole ethanol liquid (take by weighing the 0.1g carbazole, add dehydrated alcohol 100ml solution) 0.1ml in each test tube, fully behind the mixing, place room temperature to place 2h, respectively with the light absorption value of each sample liquid of spectrophotometric determination 530nm place.Draw glucuronic acid standard curve (degree of fitting is more than or equal to 0.995) according to concentration of standard solution and light absorption value, see shown in Figure 1ly, curvilinear equation is y=0.0384x+0.1383, R
2=0.9981.
(2) get the fibroin albumen hyaluronic acid pluralgel 0.5g that embodiment 1 ~ 3 prepares respectively; (vigor of hyaluronidase is 838U/mg, and sigma), 37 ℃ are incubated degraded 65h to add 300U/ml hyaluronidase liquid 2ml respectively; Using pH value more respectively is that 7.4 PBS buffer is settled to 10ml with above-mentioned reactant liquor; The reactant liquor of respectively getting behind the 1ml standardize solution adds dehydrated alcohol 4ml, and at the centrifugal 15min of 10000r/min, getting supernatant 2ml respectively, to add pH value be 7.4 PBS buffer 3ml; As sample liquid, one group of parallel control.
Get the 0.5ml sample liquid respectively and place test tube, with the 0.5ml distilled water as contrast.Sample cell and control tube are placed ice-water bath together, in every pipe, add sodium tetraborate sulphuric acid 2.5ml lentamente respectively with glass pipet, the limit edged shakes up (to be noted; Need abundant mixing); Take out after placing boiling water bath to boil 20min, be cooled to room temperature (can ice bath, quicken cooling).The carbazole ethanol liquid 0.1ml that adds mass concentration 0.1% in each test tube fully behind the mixing, places room temperature to place 2h, with the light absorption value of spectrophotometric determination 530nm place sample liquid.Discharge glucuronic acid after the hyaluronate sodium degraded in the fibroin albumen hyaluronic acid pluralgel; The glucuronic acid that the multiform more of degraded becomes is just many more; Equation of linear regression according to the glucuronic acid standard curve; Check in the glucal acid concentration of sample liquid, and then obtain the external anti-degradation capability of fibroin albumen hyaluronic acid pluralgel, result such as table 2 are with shown in Figure 2.Hyaluronidase enzymolysis situation computational methods are:
The equal concentration (ug/ml) of the glucuronic acid in the dilute sample that CS-linear regression draws; The weight (1g=1ml) of Ws-weighing gel;
401.3=the molecular weight of disaccharidase;
194.1=D-the molecular weight of glucuronic acid;
The N=extension rate;
Can find out that along with the continuous increase of fibroin albumen addition, the resistance to enzymolysis ability of gel increases, the time of external maintenance increases.
The external anti-degradation capability of table 2 fibroin albumen hyaluronic acid pluralgel
| Sample | The glucal acid concentration | The hyaluronidase enzymolysis |
| Embodiment 1 | 56.76ug/ml | 29.34mg/ml |
| Embodiment 2 | 37.24ug/ml | 19.25mg/ml |
| Embodiment 3 | 22.31ug/ml | 11.53mg/ml |
Embodiment 6: cell toxicity test
Cell toxicity test is in order to investigate the safety of fibroin albumen hyaluronic acid pluralgel.
Specific embodiments is following:
The cell culture fluid final concentration consists of: 10%FBS, and 90%H-DMEM, solvent are ultra-pure water, pH value is 7.0 ~ 7.2.
The test cell is the L929 fibroblast, and test uses cell to be the eugonic cell of the 48h-72h that goes down to posterity.
(1) hyaluronic acid derivatives that the fibroin albumen hyaluronic acid pluralgel of embodiment 1 ~ 3 preparation and embodiment 4 is prepared is mixed with the mixed liquor of 1mg/ml with cell culture fluid, places 37 ± 1 ℃ of following lixiviate 24 ± 2h respectively, and is centrifugal, gets lixiviating solution (supernatant).
(2) with cell culture fluid preparation 1 * 10
4Individual/ml L929 (available from Shanghai Inst. of Life Science, CAS cell resource center) cell suspension divides and annotates in 96 well culture plates, establishes blank, positive control and test group, and every group at least 6 hole, every hole meet 100 μ L.Place to contain 5% (V/V) carbon dioxide air, cultivate 24h in 37 ℃ the incubator.Discard former culture medium behind the 24h, the blank group adds fresh cell culture fluid, and positive controls adds positive control solution; Be dimethyl sulfoxide (DMSO); Test group adds the lixiviating solution of step (1) preparation, and every hole 100 μ L put into above-mentioned culture environment and keep 72h.
(3) adding 20 μ L mass concentrations in every hole of step (2) culture plate is the MTT solution of 5g/L, continues to cultivate under the same conditions 4h, inhales and goes stock solution, adds the dimethyl sulfoxide (DMSO) in 150 μ L/ holes.Concussion 10min, ELIASA (infinite200 Tecan) goes up the absorbance of test 570nm wavelength, and calculates the relative propagation degree (RGR) of cell according to formula (1), carries out Cytotoxic evaluation of result, and the result sees shown in the table 3:
RGR (%)=experimental group mean light absorbency value/blank group mean light absorbency value formula (1)
The relative propagation degree of table 3 cell
Can find out that from The above results degree of propagation is all more than 90% relatively for the cell in vitro of four kinds of materials, material toxicity is minimum or almost non-toxic.
The injection testing of embodiment 7:SD rat
The in vivo test of SD rat, the effective biocompatibility of testimonial material, if good biocompatibility, tissue inflammation reaction is little; Otherwise biocompatibility is poor, and tissue inflammation reaction is big.
Through contrast fibroin albumen hyaluronic acid pluralgel and the simple intravital inflammatory reaction situation of cross-linking hyaluronic acid gel, investigate the biocompatibility of material.
The practical implementation case is following:
The SD rat of 12 200-250g of anesthesia is the hyaluronic acid derivatives of the fibroin albumen hyaluronic acid pluralgel of embodiment 1 ~ 3 preparation, embodiment 4 preparations and pH value that 7.4 PBS buffer (contrast) is expelled to the SD subcutaneous rat respectively, 3 every group; The Intradermal administered dose is 0.5cc; 6 points of each animal injection are centrage with the back, left-right symmetric; 3 points of every side are spaced apart 2cm between the injection site.In 2,4,6,8 weeks, get 1 SD rat respectively for every group, the tissue at each position is taken a sample, make the fixedly tissue slice of embedding, carry out HE dyeing, and examine under a microscope tissue reaction, the result sees Fig. 3 and shown in Figure 4.
A figure is the tissue slice microgram after the fibroin albumen hyaluronic acid pluralgel of embodiment 1 preparation implanted for two weeks in vivo among Fig. 3; B figure is the tissue slice microgram after the fibroin albumen hyaluronic acid pluralgel of embodiment 1 preparation implanted for eight weeks in vivo, and arrow indication position is the delivery position of gel rubber material and tissue.Fig. 3 can find out that As time goes on, the inflammatory reaction that the implantation sample causes is in vivo slowly diminishing, and after eight weeks, does not almost have the inflammatory reaction phenomenon.
Fig. 4 is implanted into the situation in 2 weeks through observing four kinds of sample bodies, investigates the inflammatory reaction situation of different samples.Wherein, A is the HE dyeing picture that embodiment 1 sample (fibroin albumen hyaluronic acid pluralgel) body was implanted into for two weeks; B is the HE dyeing picture that embodiment 2 samples (fibroin albumen hyaluronic acid pluralgel) body was implanted into for two weeks; C is the HE dyeing picture that embodiment 3 samples (fibroin albumen hyaluronic acid pluralgel) body was implanted into for two weeks; D is the HE dyeing picture that embodiment 4 samples (hyaluronic acid derivatives) body was implanted into for two weeks, and E is the HE dyeing picture after check sample (PBS) body was implanted into for two weeks.As can be seen from Figure 4, four groups of samples are compared with PBS, and body is implanted into and certain inflammatory reaction can occur; A, B, three samples of C are compared with D; The inflammatory reaction situation does not have tangible difference, and (addition from A to the C fibroin albumen is 1/30,1/20 of a hyaluronate sodium quality along with fibroin albumen concentration constantly increases; 1/10); The biocompatibility of material obviously changes, so the adding of fibroin albumen can't influence the biocompatibility of material.
Claims (10)
1. injection fibroin albumen hyaluronic acid pluralgel is characterized in that said pluralgel is made up of following quality proportion raw material: 1 part of fibroin albumen microsphere, 1 ~ 50 part of hyaluronate and 0.08 ~ 4 part of cross-linking agent; Said fibroin albumen microsphere be silk fibroin protein solution with mass concentration 5% after leaving standstill gel under 25 ℃, homogenizing 5min sieves under the 24000rpm/min condition, collects the fibroin albumen microsphere of 200 ~ 325 mesh sieves; Said cross-linking agent is the mixing of one or more arbitrary proportions in divinylsulfone, Ethylene glycol diglycidyl ether, butanediol diglycidyl ether or the polypropylene glycol diglycidyl ether.
2. injection fibroin albumen hyaluronic acid pluralgel according to claim 1 is characterized in that said hyaluronate is the mixing of one or more arbitrary proportions in hyaluronate sodium, potassium hyaluronate, the hyaluronic acid magnesium.
3. injection fibroin albumen hyaluronic acid pluralgel according to claim 1 is characterized in that said pluralgel is made up of following quality proportion raw material: 1 part of fibroin albumen microsphere, 10 ~ 30 parts of hyaluronates and 0.8 ~ 2.4 part of cross-linking agent.
4. injection fibroin albumen hyaluronic acid pluralgel according to claim 1 is characterized in that said pluralgel is made up of following quality proportion raw material: 1 part of fibroin albumen microsphere, 30 parts of hyaluronates and 2.4 parts of cross-linking agent.
5. injection fibroin albumen hyaluronic acid pluralgel according to claim 1 is characterized in that said pluralgel is made up of following quality proportion raw material: 1 part of fibroin albumen microsphere, 20 parts of hyaluronates and 1.6 parts of cross-linking agent.
6. injection fibroin albumen hyaluronic acid pluralgel according to claim 1 is characterized in that said pluralgel is made up of following quality proportion raw material: 1 part of fibroin albumen microsphere, 10 parts of hyaluronates and 0.8 part of cross-linking agent.
7. the method for preparing of injection fibroin albumen hyaluronic acid pluralgel according to claim 1 is characterized in that said method is: the preparation of (1) fibroin albumen microsphere: Bombyxmori Linnaeus silkworm silk is joined in the aqueous sodium carbonate of mass concentration 0.2% 96 ~ 100 ℃ of water-bath 60min; Filter; Get filter cake and repeat water-bath operation 3 times, get that the lithium bromide water solution with 9mol/L dissolves behind the last filtering filtration cakes torrefaction, at 60 ℃ of water-bath 6h; Centrifugal 5min under the condition of 8000rpm then; Collect supernatant, using molecular cut off is that 8000 ~ 14000 bag filter is at room temperature dialysed, and is dialysis solution with the purified water; The silk fibroin protein solution that the trapped fluid water is mixed with mass concentration 5% is got in magnetic agitation dialysis 3 days; After leaving standstill gel under 25 ℃, homogenizing 5min sieves under the 24000rpm/min condition, collects the fibroin albumen microgranule of 200 ~ 325 mesh sieves with the silk fibroin protein solution of mass concentration 5%; The volumetric usage of said aqueous sodium carbonate is counted 67.5 ~ 125ml/g with the Bombyxmori Linnaeus silk quality, and the volumetric usage of said lithium bromide water solution is counted 5 ~ 6.25ml/g with the Bombyxmori Linnaeus silk quality; (2) preparation of injection fibroin albumen hyaluronic acid pluralgel: the hyaluronic acid saline solution that the hyaluronate of formula ratio is mixed with 0.1g/ml with the sodium hydrate aqueous solution of mass concentration 1%; Fibroin albumen microgranule with formula ratio joins abundant mixing in the above-mentioned hyaluronic acid saline solution then; Add formula ratio cross-linking agent mixing again, leave standstill 2 ~ 8h under 25 ~ 60 ℃ of conditions, form fibroin albumen hyaluronic acid composite gel material; It is 8000 ~ 14000 bag filter that fibroin albumen hyaluronic acid composite gel material is placed molecular cut off; With pH is that 7.4 PBS buffer is that dialysis solution carries out dialysis treatment, gets the composite gel material after the dialysis, homogenizing 5 ~ 15min under the 24000rpm/min condition; Cross 60 mesh sieves, collection cut size is that the granule of 60 mesh sieves is said injection fibroin albumen hyaluronic acid pluralgel; Said hyaluronate is the mixing of one or more arbitrary proportions in hyaluronate sodium, potassium hyaluronate, the hyaluronic acid magnesium, and said cross-linking agent is the mixing of one or more arbitrary proportions in divinylsulfone, Ethylene glycol diglycidyl ether, butanediol diglycidyl ether or the polypropylene glycol diglycidyl ether.
8. like the method for preparing of the said injection fibroin albumen of claim 7 hyaluronic acid pluralgel, it is characterized in that the said reaction temperature of step (2) is 30 ~ 40 ℃.
9. like the method for preparing of the said injection fibroin albumen of claim 7 hyaluronic acid pluralgel, it is characterized in that step (2) adds behind the cross-linking agent mixing that time of repose is 4 ~ 6h under 30 ~ 40 ℃ of conditions again.
10. the application of injection fibroin albumen hyaluronic acid pluralgel in the preparation soft tissue filling material according to claim 1.
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