CN102836465B - 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 Medical Cosmetic Collagen Injection packing material, particularly a kind of injection fibroin albumen hyaluronic acid pluralgel and preparation method and the application in soft tissue filling material.
(2) background technology
At present tissue repairing, tissue strengthen in, the rapid development of non-operative treatment, proportion has exceeded 80%; Because local injection belongs to minimally-invasive treatment method, only need, by some drugs and the lax soft tissue part of chemicals injecting structure, improve partial relaxation degree, improve the ability of local proof pressure, thereby make its normal function of this organized renewing or outward appearance.
Along with scientific and technical development, tissue filling material is also more and more, they can be divided into biomaterial class and cell injection product class according to its composition and biological property.
Adopting cell injection is the Therapeutic Method that has application prospect, and cell can synthesize and extracellular matrix secretion, can promote the synthetic of collagen molecules, recovers defective tissue.But because cell products mostly is liquid, be expelled to and be difficult to control it after local organization and distribute, can not plastotype, affect the effect of tissue repairing, enhancing.
The biomaterial for tissue repairing, enhancing class of having reported 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-Glucose aldehydic acid mutually combine, owing to not having kind and internal organs specificity.Hyaluronic acid has special hydrodynamic performance, and this makes it have height viscoelasticity, plasticity, permeability and good biocompatibility.It also has the tissue adhesion of preventing, greatly reduces postoperative complication, meanwhile, has promotion wound healing, reduces the advantage of cicatrix.After being implanted in human body, do not cause allosome rejection and anaphylaxis.
Natural ectogenic hyaluronan molecule amount is greatly about 100,000 ~ 5,000,000 Dalton, and retention time in vivo about about 3 ~ 15 days, has limited its application in some field greatly to a great extent.
Patent CN1590444A adopts hyaluronic acid (HA) to react 2-24 hour with cross-linking agent (BDDE) by proper proportion, forms hyaluronic acid derivatives.Single hyaluronic acid derivatives, as soft tissue filling material, will decompose absorption because of metabolism after a while, does not reach the object of long-term filling.
The method that patent CN101502676A adopts is that polymethyl methacrylate (PMMA) microsphere and cross-linked hyaluronic acid gel microgranule are mixed.Due to the existence of PMMA microsphere, make product retaining in when injected organism tissue there is persistency.But because PMMA is a kind of high molecular polymer, can produce foreign body to local organization stimulates, biocompatibility is relatively poor.
Research shows, silkworm silk is made up of approximately 75% fibroin and approximately 25% sericin, it be silkworm at silk glands endocrine native protein out, formed by 18 seed amino acids, mainly formed by the Gly-Ala-Gly-Ala-Ser polyembryony unit repeating, have certain similar to human collagen albumen.Its avirulence, nonirritant, be that a kind of degraded is slow, and metastable biomaterial, is widely used in the fields such as biomaterial subject, organizational project and drug release.
There is researcher to adopt the methods such as supersonic induced or polymer electrostatic interaction to prepare fibroin albumen-hyaluronic acid pluralgel.Compared with simple hyaluronic acid derivatives, greatly extend the degradation time of gel.The pluralgel that so adopts said method to prepare, only depends on simple fibroin albumen, or physical action between fibroin albumen and hyaluronic acid, and hyaluronic acid contents is less, and the water absorption of pluralgel is poor.
Therefore, find a kind of method of preparing fibroin albumen and hyaluronic acid pluralgel, can make material tool good biocompatibility, and have good water absorption, degradation speed slowly concurrently.Therefore seek a kind of long fibroin albumen and hyaluronic acid composite of retention time in good biocompatibility, water absorption, body that have concurrently, tissue repairing, reinforcing material that can be permanently effective, 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, the method of hyaluronic acid, fibroin albumen composite material is prepared in employing, provide a kind of good biocompatibility, mechanical property and good water absorption of having concurrently, tissue repairing, reinforcing material that can be permanently effective.
The technical solution used in the present invention is:
A kind of injection fibroin albumen hyaluronic acid pluralgel, described pluralgel is made up of the raw material of following quality proportioning: 1 part of fibroin albumen microsphere, 0.08 ~ 4 part of 1 ~ 50 part of hyaluronate and cross-linking agent; Described fibroin albumen microsphere is that the silk fibroin protein solution of mass concentration 5% is left standstill after gel at 25 DEG C, and under 24000rpm/min condition, homogenizing 5min, sieves, and collects the fibroin albumen microsphere of 200 ~ 325 mesh sieves; Described 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 polypropylene glycol diglycidyl ether, most preferably butanediol diglycidyl ether.
Described hyaluronate is preferably the mixing of one or more arbitrary proportions in hyaluronate sodium, potassium hyaluronate, hyaluronic acid magnesium, more preferably hyaluronate sodium.
Further, described pluralgel is made up of the raw material of following quality proportioning: 1 part of fibroin albumen microsphere, 0.8 ~ 2.4 part of 10 ~ 30 parts of hyaluronates and cross-linking agent.
Further, described pluralgel is made up of the raw material of following quality proportioning: 1 part of fibroin albumen microsphere, 2.4 parts of 30 parts of hyaluronates and cross-linking agent.
Further, described pluralgel is made up of the raw material of following quality proportioning: 1 part of fibroin albumen microsphere, 1.6 parts of 20 parts of hyaluronates and cross-linking agent.
Further, described pluralgel is made up of the raw material of following quality proportioning: 1 part of fibroin albumen microsphere, 0.8 part of 10 parts of hyaluronates and cross-linking agent.
Further, the preparation method of injection fibroin albumen hyaluronic acid pluralgel of the present 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 DEG C of water-bath 60min, filter, get filter cake and repeat water-bath operation 3 times, get after the filtration cakes torrefaction of last filtration and dissolve with the lithium bromide water solution of 9mol/L, at 60 DEG C of water-bath 6h, then centrifugal 5min under 8000rpm condition, collect supernatant, at room temperature dialyse with the bag filter that molecular cut off (MW) is 8000 ~ 14000, taking purified water as dialysis solution, magnetic agitation dialysis 3 days, get the silk fibroin protein solution that trapped fluid water is mixed with mass concentration 5% (get a certain amount of described trapped fluid in 80 DEG C of baking ovens dry 4h to constant weight, calculate the water content of trapped fluid, and then trapped fluid water is mixed with to the silk fibroin protein solution of mass concentration 5%), the silk fibroin protein solution of mass concentration 5% is left standstill after gel at 25 DEG C, and under 24000rpm/min condition, homogenizing 5min, sieves, and collects the fibroin albumen microgranule of 200 ~ 325 mesh sieves, the volumetric usage of described aqueous sodium carbonate is counted the preferred 67.5ml/g of 67.5 ~ 125ml/g(with Bombyxmori Linnaeus silk quality), the volumetric usage of described lithium bromide water solution is counted 5 ~ 6.25ml/g with 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 to 0.1g/ml with the sodium hydrate aqueous solution of mass concentration 1%, then the fibroin albumen microgranule of formula ratio is joined in above-mentioned hyaluronic acid saline solution and fully mixed, add again formula ratio cross-linking agent to mix, under 25 ~ 60 DEG C of conditions, leave standstill 2 ~ 8h, form fibroin albumen hyaluronic acid composite gel material, fibroin albumen hyaluronic acid composite gel material is placed in to the bag filter of molecular cut off (MW) 8000 ~ 14000, PBS buffer taking pH as 7.4 carries out dialysis treatment as dialysis solution, get composite gel material (the being trapped fluid) homogenizing 5 ~ 15min under 24000rpm/min condition after dialysis treatment, cross 60 mesh sieves, collection cut size is that the granule of 60 mesh sieves is described injection fibroin albumen hyaluronic acid pluralgel, described hyaluronate is the mixing of one or more arbitrary proportions in hyaluronate sodium, potassium hyaluronate, hyaluronic acid magnesium, preferably clear matter acid sodium, described cross-linking agent is the mixing of one or more arbitrary proportions in divinylsulfone, Ethylene glycol diglycidyl ether, butanediol diglycidyl ether or polypropylene glycol diglycidyl ether, preferably butanediol diglycidyl ether.
Further, after step (2) adds cross-linking agent to mix, preferably under 30 ~ 40 DEG C of conditions, leave standstill.
Further, after step (2) adds cross-linking agent to mix, preferably under 4 ~ 6h condition, leave standstill.
The advantages such as the invention provides the application of described injection fibroin albumen hyaluronic acid pluralgel in soft tissue filling material, it has uniform particle diameter, is easy to injection, compared with simple HA injected gel, and retention time is long.
Described soft tissue filling material refers to micro-shaping packing materials such as face crinkle-removing.
The average particle size range of fibroin albumen microsphere is 1 ~ 120 μ m, preferably 20 ~ 100 μ m, and more preferably 50 ~ 75 μ m, because fibroin albumen microsphere is 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 sour simple crosslinked gel phase ratio of conventional transparent matter, fibroin albumen hyaluronic acid pluralgel In Vitro Anti enzymolysis ability significantly improves, the evidence of In Vitro Anti enzymolysis, at same enzyme liquid, under same time effect, hyaluronic In Vitro Anti enzymolysis ability in fibroin albumen hyaluronic acid pluralgel increases along with the increase of fibroin albumen granule, can find thus, adding of fibroin albumen granule, effectively slow down the degradation speed of pluralgel, extend it in tissue repairing, the action time of filling, improved the effectiveness of material;
2) the present invention is compared with simple fibroin albumen granular materials, and HA adds, and has increased the water absorption of material, improves lubricity and the water storage function of material, can significantly increase the filling effect of material;
3) the present invention and simple physical method silk union fibroin granule and HA Particle Phase ratio, because fibroin albumen granule is wrapped in HA gel particle, both mixing are more even, 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) brief description of the drawings
Fig. 1 glucuronic acid standard curve;
Fig. 2 fibroin albumen hyaluronic acid pluralgel In Vitro Anti degradation capability: the fibroin albumen hyaluronic acid pluralgel that sample 1 is prepared for embodiment 1, the fibroin albumen hyaluronic acid pluralgel that sample 2 is prepared for embodiment 2, the fibroin albumen hyaluronic acid pluralgel that sample 3 is prepared for embodiment 3;
Fig. 3 fibroin albumen hyaluronic acid pluralgel is implanted the tissue slice microgram after two weeks in vivo, A is that fibroin albumen hyaluronic acid pluralgel prepared by embodiment 1 is implanted the tissue slice microgram after two weeks in vivo, and B is that fibroin albumen hyaluronic acid pluralgel prepared by 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 that embodiment 1 ~ 4 prepares fibroin albumen hyaluronic acid pluralgel and hyaluronic acid derivatives body is implanted into the HE dyeing picture of two weeks, and E is that check sample (PBS) body is implanted into the HE dyeing picture after two weeks.
(5) detailed description of the invention
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%, 96 ~ 100 DEG C of water-bath 60min, filter, get filter cake and repeat water-bath 3 times, the filter cake of getting last filtration slowly dissolves with the lithium bromide water solution 200ml of 9mol/L afterwards 60 DEG C of oven dry, at 60 DEG C of water-bath 6h, then the centrifugal 5min of 8000rpm, get supernatant, pack the supernatant after centrifugal treating into bag filter (MW8, 000 ~ 14, 000), taking purified water as dialysis solution, magnetic agitation dialysis three days, get trapped fluid, and to be mixed with mass concentration by purified water be 5% silk fibroin protein solution.
Under 25 DEG C of conditions, above-mentioned 5% silk fibroin protein solution is left standstill and forms white gels material 5g, by homogenizer (T25 for gel rubber material, IKA) under 24000rpm/min condition after homogenizing 5min, obtain the Silk fibroin gel granule 5g that diameter is different, filter, collected the fibroin albumen granule 3.5g of 200 mesh sieves, fibroin albumen particle drying to the constant weight that takes a small amount of collection is determined the water content of fibroin albumen granule, and definite fibroin albumen granular mass concentration is 3.23%.
(2) fibroin albumen hyaluronic acid pluralgel: 0.7g hyaluronate sodium is dissolved in 7ml mass concentration 1% sodium hydroxide solution and makes 0.1g/ml sodium hyaluronate solution, and to the fibroin albumen granule 0.712g(0.023/0.0323=0.712g that adds in sodium hyaluronate solution step (1) to obtain, fibroin albumen granule dry weight is 0.023g), after fully mixing, add 56 μ l(0.056g) BDDE(1, 4-butanediol diglycidyl ether), after mix homogeneously, be placed under 40 DEG C of conditions and leave standstill and be cross-linked for 4 hours, can form fibroin albumen-HA composite gel material.
Above-mentioned fibroin albumen-HA composite gel material is joined to MW8,000-14, in 000 bag filter, the PBS buffer taking pH value as 7.4, as dialysis solution, carries out dialysis treatment under magnetic agitation, by composite gel material after dialysis treatment with homogenizer under 24000rpm/min condition after homogenizing 10min, be placed in extruding in syringe and make gel pass through 60 mesh sieves, collect composite gel particle, after 120 DEG C of high temperature and high pressure steam sterilizing 15min, carry out aseptic subpackagedly, pack in disposable syringe.The fibroin albumen hyaluronic acid pluralgel 35g that obtains injection, can be used as hypodermic products.Get the mass ratio dilutions (proportioning is shown in Table 1) such as fibroin albumen hyaluronic acid pluralgel distilled water work, after mix homogeneously, room temperature (25 DEG C) leaves standstill half an hour, measure the pH value of solution, each sample in triplicate, and is averaged as the pH value of sample, the results are shown in Table shown in 1.
(1) according to method embodiment 1 step (1) Suo Shu, prepared the fibroin albumen granule 3.5g of 200 mesh sieves, mass concentration is 3.23%.
(2) 0.7g hyaluronate sodium is dissolved in 7ml mass concentration 1% sodium hydroxide solution and makes 0.1g/ml sodium hyaluronate solution, and to the fibroin albumen granule 1.0836g(0.035/0.0323=1.0836g that adds in hyaluronic acid solution step (1) to obtain), after fully mixing, add the BDDE of 56 μ l, after mix homogeneously, be placed under 40 DEG C of conditions and leave standstill and be cross-linked for 6 hours, form fibroin albumen hyaluronic acid composite gel material.
Above-mentioned fibroin albumen hyaluronic acid composite gel material is carried out to dialysis treatment, and other operates with embodiment 1, obtains the fibroin albumen hyaluronic acid pluralgel 35g of injection, can be used as tissue filling injection product.Get the mass ratio dilutions (proportioning is shown in Table 1) such as fibroin albumen hyaluronic acid pluralgel distilled water work, after mix homogeneously, room temperature (25 DEG C) leaves standstill half an hour, measure the pH value of solution, each sample in triplicate, and is averaged as the pH value of sample, the results are shown in Table shown in 1.
Embodiment 3: fibroin albumen hyaluronic acid pluralgel
(1) according to method embodiment 1 step (1) Suo Shu, prepared the fibroin albumen granule 3.5g of 200 mesh sieves, mass concentration is 3.23%.
(2) 0.7g hyaluronate sodium is dissolved in to the sodium hyaluronate solution of making 0.1g/ml in 7ml mass concentration 1% sodium hydroxide solution, and add 2.167g(0.07/0.0323=2.167g in sodium hyaluronate solution) fibroin albumen granule, after fully mixing, add the BDDE of 56 μ l, after mix homogeneously, be placed under 40 DEG C of conditions and leave standstill and be cross-linked for 5 hours, form fibroin albumen hyaluronic acid composite gel material.
Fibroin albumen hyaluronic acid composite gel material is carried out to dialysis treatment, and other operates with embodiment 1, obtains the fibroin albumen hyaluronic acid pluralgel 35g of injection, can be used as subcutaneous filling injection product.Get the mass ratio dilutions (proportioning is shown in Table 1) such as fibroin albumen hyaluronic acid pluralgel distilled water work, after mix homogeneously, room temperature (25 DEG C) leaves standstill half an hour, measure the pH value of solution, each sample in triplicate, and is averaged as the pH value of sample, the results are shown in Table shown in 1.
Embodiment 4: the preparation of hyaluronic acid derivatives
0.7g hyaluronate sodium is dissolved in to the sodium hyaluronate solution of making 0.1g/ml in 7ml mass concentration 1% sodium hydroxide solution, after fully mixing, add 56 μ l BDDE, after mix homogeneously, be placed under 40 DEG C of conditions and leave standstill and be cross-linked for 5 hours, can obtain hyaluronic acid derivatives.
Hyaluronic acid derivatives is carried out after dialysis treatment, and other operates with embodiment 1, obtains the hyaluronic acid derivatives 35g of injection.Get the mass ratio dilutions (proportioning is shown in Table 1) such as fibroin albumen hyaluronic acid pluralgel distilled water work, after mix homogeneously, room temperature (25 DEG C) leaves standstill half an hour, measure the pH value of solution, each sample in triplicate, and is averaged as the pH value of sample, the results are shown in Table shown in 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: In Vitro Anti degradation capability test
Cross-linked-hyaluronic acid, after enzymolysis, can resolve into glucuronic acid, and glucuronic acid and the effect of carbazole reagent produce reddish violet, and the depth of color is directly proportional to the content of glucuronic acid.Can determine by light absorption value the content of glucuronic acid.
In the fibroin albumen hyaluronic acid pluralgel of making by investigation different proportion fibroin albumen addition, the number of glucuronic acid content, judge the In Vitro Anti enzymolysis ability of pluralgel, thereby judge the In Vitro Anti enzymolysis ability of pluralgel and simple cross-linking hyaluronic acid gel.
Specific embodiments is as follows:
The fibroin albumen hyaluronic acid pluralgel of embodiment 1 ~ 3 preparation is carried out to the mensuration of In Vitro Anti degradation capability.
(1) drafting of standard curve
The glucal standard acid solution of water preparation 70,60,50,40,30 μ g/ml, gets 0.5ml titer and is placed in test tube, with 0.5ml distilled water in contrast.Standard pipe and control tube are placed in to ice-water bath (0 DEG C) together, in every pipe, add respectively sodium tetraborate sulphuric acid 2.5ml lentamente with glass pipet, limit edged shakes up (to be noted, need to fully mix), be placed in after boiling water bath boils 20min and take out, be cooled to room temperature (can ice bath, accelerate cooling).In each test tube, add 0.1% carbazole ethanol (take 0.1g carbazole, add dehydrated alcohol 100ml solution) 0.1ml, after fully mixing, be placed in room temperature and place 2h, respectively with the light absorption value of the each sample liquid in 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, as shown in Figure 1, curvilinear equation is y=0.0384x+0.1383, R
2=0.9981.
(2) get respectively fibroin albumen hyaluronic acid pluralgel 0.5g prepared by embodiment 1 ~ 3, adding respectively the vigor of 300U/ml hyaluronidase liquid 2ml(hyaluronidase is 838U/mg, sigma), 37 DEG C of insulation degraded 65h, above-mentioned reactant liquor is settled to 10ml by the PBS buffer that is 7.4 with pH value respectively again, the reactant liquor of respectively getting after 1ml standardize solution adds dehydrated alcohol 4ml, at the centrifugal 15min of 10000r/min, getting respectively supernatant 2ml, to add pH value be 7.4 PBS buffer 3ml, as sample liquid, one group of parallel control.
Get respectively 0.5ml sample liquid and be placed in test tube, with 0.5ml distilled water in contrast.Sample cell and control tube are placed in to ice-water bath together, add respectively sodium tetraborate sulphuric acid 2.5ml lentamente with glass pipet in every pipe, limit edged shakes up (to be noted, need to fully mix), be placed in after boiling water bath boils 20min and take out, be cooled to room temperature (can ice bath, accelerate cooling).In each test tube, add the carbazole ethanol 0.1ml of mass concentration 0.1%, after fully mixing, be placed in room temperature and place 2h, with the light absorption value of spectrophotometric determination 530nm place sample liquid.In fibroin albumen hyaluronic acid pluralgel, after hyaluronate sodium degraded, discharge glucuronic acid, the glucuronic acid that the more multiform of degraded becomes is just more, according to the equation of linear regression of glucuronic acid standard curve, check in the glucal acid concentration of sample liquid, and then obtaining fibroin albumen hyaluronic acid pluralgel In Vitro Anti degradation capability, result is as shown in table 2 and Fig. 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; Ws-weighs the weight (1g=1ml) of gel;
401.3=the molecular weight of disaccharidase;
194.1=D-the molecular weight of glucuronic acid;
N=extension rate;
Can find out, along with the continuous increase of fibroin albumen addition, the resistance to enzymolysis ability of gel increases, and the time of external maintenance increases.
Table 2 fibroin albumen hyaluronic acid pluralgel In Vitro Anti degradation capability
| Sample | Glucal acid concentration | Hyaluronidase |
| Embodiment | ||
| 1 | 56.76ug/ml | 29.34mg/ |
| Embodiment | ||
| 2 | 37.24ug/ml | 19.25mg/ |
| Embodiment | ||
| 3 | 22.31ug/ml | 11.53mg/ml |
Embodiment 6: cell toxicity test
Cell toxicity test is the safety in order to investigate fibroin albumen hyaluronic acid pluralgel.
Specific embodiments is as follows:
Cell culture fluid final concentration consists of: 10%FBS, and 90%H-DMEM, solvent is ultra-pure water, pH value is 7.0 ~ 7.2.
Test cell is L929 fibroblast, and test is the eugonic cell of the 48h-72h that goes down to posterity with cell.
(1) hyaluronic acid derivatives cell culture fluid prepared by the fibroin albumen hyaluronic acid pluralgel of being prepared by embodiment 1 ~ 3 and embodiment 4 is mixed with the mixed liquor of 1mg/ml, is placed in respectively lixiviate 24 ± 2h at 37 ± 1 DEG C, centrifugal, gets lixiviating solution (supernatant).
(2) with cell culture fluid preparation 1 × 10
4individual/ml L929(is purchased from Shanghai Inst. of Life Science, CAS cell resource center) cell suspension, dispensing, in 96 well culture plates, is established blank, positive control and test group, every group at least 6 hole, every hole meets 100 μ L.Be placed in containing 5%(V/V) carbon dioxide air, 24h in the incubator of 37 DEG C, cultivated.After 24h, discard former culture medium, 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) add in every hole of step (2) culture plate the MTT solution that 20 μ L mass concentrations are 5g/L, continue to cultivate under the same conditions 4h, suck stock solution, add the dimethyl sulfoxide (DMSO) in 150 μ L/ holes.Concussion 10min, at the absorbance at the upper test of microplate reader (infinite200, Tecan) 570nm wavelength place, and according to the relative propagation degree (RGR) of formula (1) calculating cell, carries out Cytotoxic evaluation of result, the results are shown in Table shown in 3:
RGR (%)=experimental group mean light absorbency value/blank group mean light absorbency value formula (1)
The relative propagation degree of table 3 cell
From the above results, can find out, degree of propagation is all more than 90% relatively for the cell in vitro of four kinds of materials, and material toxicity is minimum or almost non-toxic.
The injection testing of embodiment 7:SD rat
The in vivo test of SD rat, the effectively biocompatibility of testimonial material, if good biocompatibility, tissue inflammation reaction is little; Otherwise biocompatibility is poor, tissue inflammation reaction is large.
By the inflammatory reaction situation in contrast fibroin albumen hyaluronic acid pluralgel and simple cross-linking hyaluronic acid gelinite, investigate the biocompatibility of material.
Concrete case study on implementation is as follows:
The SD rat of 12 200-250g of anesthesia, the PBS buffer (contrast) that fibroin albumen hyaluronic acid pluralgel prepared by embodiment 1 ~ 3, hyaluronic acid derivatives prepared by embodiment 4 and pH value are 7.4 is expelled to respectively SD subcutaneous rat, every group 3, Intradermal administered dose is 0.5cc, 6 points of each animal injection, line centered by back, symmetrical, 3 points of every side, are spaced apart 2cm between injection site.At 2,4,6,8 weeks, get respectively 1 SD rat for every group, the tissue at each position is sampled, make the tissue slice of fixing embedding, carry out HE dyeing, and examine under a microscope tissue reaction, the results are shown in Figure 3 and Fig. 4 shown in.
In Fig. 3, A figure is that fibroin albumen hyaluronic acid pluralgel prepared by embodiment 1 is implanted the tissue slice microgram after two weeks in vivo, B figure is that fibroin albumen hyaluronic acid pluralgel prepared by embodiment 1 is implanted the tissue slice microgram after eight weeks in vivo, and arrow indication position is the delivery position of gel rubber material and tissue.Fig. 3 can find out, As time goes on, implants the inflammatory reaction that sample causes in vivo and slowly diminishing, and after eight weeks, almost there is no inflammatory reaction phenomenon.
Fig. 4 is the situation that is implanted into 2 weeks by 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 is implanted into two weeks, B is the HE dyeing picture that embodiment 2 samples (fibroin albumen hyaluronic acid pluralgel) body is implanted into two weeks, C is the HE dyeing picture that embodiment 3 samples (fibroin albumen hyaluronic acid pluralgel) body is implanted into two weeks, D is the HE dyeing picture that embodiment 4 samples (hyaluronic acid derivatives) body is implanted into two weeks, and E is that check sample (PBS) body is implanted into the HE dyeing picture after two weeks.As can be seen from Figure 4, four groups of samples are compared with PBS, and body is implanted into and there will be certain inflammatory reaction; A, B, tri-samples of C are compared with D, inflammatory reaction situation does not have obvious difference, and (addition from A to C fibroin albumen is hyaluronate sodium quality 1/30 along with fibroin albumen concentration constantly increases, 1/20,1/10), the biocompatibility of material does not obviously change, therefore adding of fibroin albumen can't affect the biocompatibility of material.
Claims (8)
1. an injection fibroin albumen hyaluronic acid pluralgel, is characterized in that described pluralgel is made up of the raw material of following quality proportioning: 1 part of fibroin albumen microsphere, 0.08～4 part of 1～50 part of hyaluronate and cross-linking agent;
Described injection fibroin albumen hyaluronic acid pluralgel is prepared as follows: 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 DEG C of water-bath 60min, filter, get filter cake and repeat water-bath operation 3 times, get after the filtration cakes torrefaction of last filtration and dissolve with the lithium bromide water solution of 9mol/L, at 60 DEG C of water-bath 6h, then centrifugal 5min under the condition of 8000rpm, collect supernatant, at room temperature dialyse with the bag filter that molecular cut off is 8000～14000, taking purified water as dialysis solution, magnetic agitation dialysis 3 days, get the silk fibroin protein solution that trapped fluid water is mixed with mass concentration 5%, the silk fibroin protein solution of mass concentration 5% is left standstill after gel at 25 DEG C, and under 24000rpm/min condition, homogenizing 5min, sieves, and collects the fibroin albumen microgranule of 200～325 mesh sieves, the volumetric usage of described aqueous sodium carbonate is counted 67.5～125ml/g with Bombyxmori Linnaeus silk quality, and the volumetric usage of described lithium bromide water solution is counted 5～6.25ml/g with 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 to 0.1g/ml with the sodium hydrate aqueous solution of mass concentration 1%, then the fibroin albumen microgranule of formula ratio is joined in above-mentioned hyaluronic acid saline solution and fully mixed, add again formula ratio cross-linking agent to mix, under 25～60 DEG C of conditions, leave standstill 2～8h, form fibroin albumen hyaluronic acid composite gel material, it is 8000～14000 bag filter that fibroin albumen hyaluronic acid composite gel material is placed in to molecular cut off, PBS buffer taking pH as 7.4 carries out dialysis treatment as dialysis solution, get the composite gel material after dialysis, homogenizing 5～15min under 24000rpm/min condition, cross 60 mesh sieves, collection cut size is that the granule of 60 mesh sieves is described injection fibroin albumen hyaluronic acid pluralgel, described hyaluronate is the mixing of one or more arbitrary proportions in hyaluronate sodium, potassium hyaluronate, hyaluronic acid magnesium, and described cross-linking agent is the mixing of one or more arbitrary proportions in divinylsulfone, Ethylene glycol diglycidyl ether, butanediol diglycidyl ether or polypropylene glycol diglycidyl ether.
2. injection fibroin albumen hyaluronic acid pluralgel as claimed in claim 1, is characterized in that described pluralgel is made up of the raw material of following quality proportioning: 1 part of fibroin albumen microsphere, 0.8～2.4 part of 10～30 parts of hyaluronates and cross-linking agent.
3. injection fibroin albumen hyaluronic acid pluralgel as claimed in claim 1, is characterized in that described pluralgel is made up of the raw material of following quality proportioning: 1 part of fibroin albumen microsphere, 2.4 parts of 30 parts of hyaluronates and cross-linking agent.
4. injection fibroin albumen hyaluronic acid pluralgel as claimed in claim 1, is characterized in that described pluralgel is made up of the raw material of following quality proportioning: 1 part of fibroin albumen microsphere, 1.6 parts of 20 parts of hyaluronates and cross-linking agent.
5. injection fibroin albumen hyaluronic acid pluralgel as claimed in claim 1, is characterized in that described pluralgel is made up of the raw material of following quality proportioning: 1 part of fibroin albumen microsphere, 0.8 part of 10 parts of hyaluronates and cross-linking agent.
6. injection fibroin albumen hyaluronic acid pluralgel as claimed in claim 1, is characterized in that the described reaction temperature of step (2) is 30～40 DEG C.
7. injection fibroin albumen hyaluronic acid pluralgel as claimed in claim 1, is characterized in that under 30～40 DEG C of conditions, leaving standstill 4～6h after step (2) adds cross-linking agent and mixes again.
As claimed in claim 1 injection fibroin albumen hyaluronic acid pluralgel in the application of preparing in soft tissue filling material.
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| CN105985529B (en) * | 2015-03-05 | 2020-11-10 | 华中科技大学同济医学院附属协和医院 | Sericin-alginate composite hydrogel and preparation method thereof |
| US10300169B2 (en) | 2016-08-24 | 2019-05-28 | Allergan, Inc. | Co-crosslinked hyaluronic acid-silk fibroin hydrogels for improving tissue graft viability and for soft tissue augmentation |
| CN106492279A (en) * | 2016-11-04 | 2017-03-15 | 武汉纺织大学 | A kind of fast preparation method of fibroin albumen hyaluronic acid pluralgel |
| CN106589424B (en) * | 2016-12-12 | 2020-06-02 | 华熙生物科技股份有限公司 | Cross-linked hyaluronic acid gel for injection and preparation method thereof |
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| US20230190997A1 (en) * | 2017-06-26 | 2023-06-22 | Evolved By Nature, Inc. | Silk-hyaluronic acid based tissue filers and methods of using the same |
| IL299584A (en) * | 2017-06-26 | 2023-03-01 | Evolved By Nature Inc | Silk-hyaluronic acid based tissue fillers and methods of using the same |
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