CN1294994C - Injectable type collagen-based soft tissue filling material and preparation method thereof - Google Patents
Injectable type collagen-based soft tissue filling material and preparation method thereof Download PDFInfo
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- CN1294994C CN1294994C CNB2004100278973A CN200410027897A CN1294994C CN 1294994 C CN1294994 C CN 1294994C CN B2004100278973 A CNB2004100278973 A CN B2004100278973A CN 200410027897 A CN200410027897 A CN 200410027897A CN 1294994 C CN1294994 C CN 1294994C
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Abstract
The present invention discloses injection type collagen based soft tissue filling material and a preparing method thereof. The filling material is mainly composed of 0.5 to 20% of collagen, 0.1 to 10% of heparin, 0 to 1% of cell growth factors and 70 to 99.4% of buffer solution. The method for perparing the filling material comprises: soluble collagen is extracted from connective tissues of animals, and protease hydrolysis is utilized to cut off epitope antigen determinants in collagen molecules. After purification, safe and nontoxic crosslinking agents are used for crosslinking, and then, biological active constituents are added. The mixture is repeatedly kneaded into paste, sealed under the aseptic condition and stored at low temperature. The obtained filling material of the present invention has the advantages of safety, no toxicity, biological degradation and easy tolerance by human bodies.
Description
Technical field
The present invention relates to biomedical material and preparation method thereof, especially tissue reparation or packing material and preparation method thereof.
Background technology
Tissue engineering technique is a kind of biotechnology that proposes and grow up gradually in the eighties in 20th century.Its basic meaning is that cell, synthetic material or the natural material of handling, tissue and cytokine and gene therapy are combined, and is widely used in intravital tissue regeneration or external tissue construction.Therefore, tissue engineering technique is a great revolution of shaping and reconstruction surgical field.The organization engineering skin product has obtained the approval of FDA and produced in USA and sell.At present at home and abroad, the research of organizational project goods has caused that people pay close attention to widely.
Similar products in the market mainly contain three classes.The one, be the product of the commodity " Zyderm " and " Zyplast " by name of representative with the facial esthetic surgery center of U.S. Ba Feite (Parfitt Facial Cosmetic Surgery Center), be the pure collagen protein that extracts purification from Corii Bovis seu Bubali, form through the glutaraldehyde chemical crosslinking.Mainly there is the problem of several aspects in this product, comprises the threat of crazy heifer disease virus, and filling effect is not good, need the continuous several times injection, and the bounce-back rate is very high, and residual glutaraldehyde has cytotoxicity, needs strict its residual quantity of control, does not sell at home as yet at present; Two, China's prevention Liu of research institute grasps the human placental collagen protein injection liquid of kind research and development.Though this product safety is higher, has immunological rejection equally, and the placental collagen degraded is fast, needs multiple injection and reparation skin effects poor, and production cost is higher relatively; Three, the commodity of Dutch Ha Fu Biological Science Co., Ltd (Hafod Bioscience B.V.) by name " Artecoll " (Artecoll) polymethyl methacrylate (PMMA) microsphere and the composite injection of collagen, this product is used the synthesized polymer material PMMA of Nondegradable as filler, material retains in vivo for a long time, homergy to skin produces harmful effect, and the potential possibility that causes skin generation canceration is arranged, do not meet people's consumer psychology.
Summary of the invention
Purpose of the present invention just provides a kind of safety non-toxic, and is biodegradable, easily for human body tolerance and possess injectable type collagen base soft tissue filling material of induced tissue reproducing characteristic and preparation method thereof.
For reaching above-mentioned purpose, collagen base soft tissue filling material of the present invention mainly is made of following component:
Soluble collagen 0.5～20%, heparin 0.1～10%, cell growth factor 0～1%, pH value is 6.8～7.2 buffer 70～99.4%.
As a preferred version, collagen base soft tissue filling material mainly is made of following component:
Soluble collagen 2～10%, heparin 0.5～5%, cell growth factor bFGF 0.0001%～0.001%, the phosphate buffer 85～97.1% of pH7.2, local anesthetic lignocaine 0.3%.
The method for preparing above-mentioned packing material is as follows: extract soluble collagen from animal connective tissue, utilize the antigen decision family in the protease hydrolysis excision tropocollagen molecule, after the purification process, adopt the cross-linking agent of safety non-toxic crosslinked, add biological active component then, mixture is kneaded into paste repeatedly, sealed under aseptic conditions and low temperature storage.
Described animal connective tissue comprises skin, cartilage and the tendon tissue etc. that derive from pig, cattle or sheep etc.
Protease is pepsin, trypsin or bromelain etc.
Described purification process is to remove wherein inorganic salt and small molecular weight impurity by dialysis or ultrafiltration membrane treatment.
Described cross-linking agent is carbodiimides or chitose.When adopting carbodiimides, the final concentration of its adding is 0.5～5%; When adopting chitose, the final concentration of its adding is 1%～20%.
Described biological active component is one or more in cytokine composition, heparin, mucopolysaccharide, chondroitin sulfate, dermatan sulfate, hyaluronic acid or the chitose.
As improvement of the present invention, in the said method, after the soluble collagen lyophilization, add cross-linking agent, crosslinked carry out 0.1～4 hour after, handled 1～48 hour 100～120 ℃ of heat treated further crosslinked and sterilization in 0～72 hour under vacuum condition then-80～0 ℃ of following lyophilization.
Adopt prepared material of the present invention and preparation method thereof to have the following advantages:
1. with the native protein macromolecule that derives from animal connective tissue as host material, safety non-toxic, biodegradable, be the human body tolerance easily;
2. material is handled through enzyme, and corresponding antigen decision family significantly reduces the antigenicity of material in the excision molecule;
3. the degradation time of material can be realized artificial adjusting easily;
4. the easy Carbodiimides cross-linking agent of removing of the crosslinked employing safety non-toxic of material replaces glutaraldehyde and formaldehyde, can react under the condition of gentleness, and cross-linking effect is good;
5. material compound cells somatomedin can induce the cell in the human body skin to quicken the corresponding extracellular matrix of propagation justacrine, realizes from the body reparation;
6. use this material and can realize the permanent reparation of soft tissues such as skin, not bounce-back.
The present invention has abandoned the defective of existing skin repair usefulness and other medical tissue packing material, according to the ultimate principle of tissue engineering technique, in conjunction with novel biological tissue's crosslinking technological, prepares novel medical packing material.The packing material of gained of the present invention is expelled to the human body skin skin corium, damaged and finally induce the fibroblast of skin corium to quicken the secretory cell epimatrix to fill corium, realize the purpose of guide tissue regeneration, to eliminate skin wrinkle and to subside, make skin recover full and elasticity, perfect used for reparing skin defect.This packing material can also be used for as therapeutic use such as tissue filling material treatment prostate excision postoperative urinary incontinences.Technology of the present invention is simple, and production cost is low.
The specific embodiment
Embodiment one:
The method for preparing packing material of the present invention is as follows successively:
1) get the fresh pig tendon tissue 1kg that butchers, clean, chopping, 10 liters of 4.5mol/l NaCl solution soaking 4 hours, then Tris-hydrochlorate of 0.05mol/l (Tris-HCl) (pH=7.5) buffer soaked 4～6 hours for 10 liters;
2) with final concentration be the mixed liquid dipping 2～12 hours of the glucose of 0.5mol/l and the NaCl that final concentration is 2mol/l;
3) acetum with 0.1mol/l soaked 4～6 hours, and tissue mashing machine smashs to pieces;
4) slurry uses 5 liters of Tris-HCl (pH=7.5) buffer to soak again 4～6 hours;
5) centrifugal treating, sediment separate out, with deposit by 1: 50 mass ratio distilled water diluting, dispersed with stirring, with about pure acetic acid regulation system pH value to 3.0, adding simultaneously accounts for 3% pepsin of deposit quality, 10 ℃ of stirring reactions 24 hours;
6) in reaction system, add disodium EDTA (EDTA), make its concentration reach 1000ppm, continue to stir 0.5 hour;
7) reactant liquor is handled at 4～10 ℃ of frozen centrifugations, removed deposit, add saturated sodium hydroxide solution in the clear liquid and be neutralized to neutrality, and then add sodium chloride, reach 2mol/l until its concentration; Slowly stir, leave standstill the 10～24h that saltouts then;
8) 4～10 ℃ of frozen centrifugation collecting precipitations with distill water dialysis 24～48 hours, changed one time water every 4 hours, detect less than Cl to silver nitrate reagent
-Till;
9) after 4～10 ℃ of frozen centrifugations were handled, precipitate became collagen sponge, about 120g-40～-10 ℃ of lyophilizations;
10) collagen sponge of getting about 1g is soaked in 50ml and contains in the alcoholic solution of 2-morpholino ethylsulfonic acid (MES) of 50mmol/l, and pH is 5.4, soaks 30min under the room temperature;
11) add N-(3-dimethylamino-propyl)-N '-ethyl carbodiimides (EDC) and N-hydroxy-succinamide (NHS), make EDC: NHS: Coll-COOH=7.0: 2.8: 1, as cross-linking agent (Coll-COOH represents the carboxyl in the tropocollagen molecule), crosslinked 1～4h under the room temperature;
12) after the phosphate buffer flushing with pH=9.0, after the NaCl solution soaking of reuse 2mol/l, distilled water wash;
13) product is once more-40～-10 ℃ of lyophilizations, 100～120 ℃ of further crosslinked and sterilizations of heat treated 1h under vacuum condition then;
14) under the aseptic condition product is added the sterile phosphate buffer solution of 20ml pH=7.2, make its abundant water absorption and swelling, knead into paste, add the 0.5g heparin then, 0.1g dermatan sulfate, 0.3% lignocaine, fully mix, under the aseptic condition paste is sealed in the disposable syringe, every pipe encapsulation 1～2ml preserves down for 10 ℃.
The composition of gained packing material is: collagen 2～10%, heparin 0.5～5%, cell growth factor bFGF0.0001%～0.001%, the phosphate buffer 85～97.1% of pH7.2, local anesthetic lignocaine 0.3%.
Embodiment two:
The method for preparing packing material of the present invention is as follows successively:
1) get the fresh calf-skin skin tissue 1kg that butchers, clean, chopping, 10 liter 10% (quality and volume ratio, soaking in sodium carbonate solution g/ml) be after 4 hours, 4.5mol/l NaCl solution soaking 4 hours;
2) the final concentration glucose solution and the 2mol/lNaCl solution soaking that are respectively 0.5mol/l spent the night;
3) acetum with 0.1mol/l soaked 4～6 hours, and tissue mashing machine smashs to pieces;
4) slurry uses 5 liters of Tris-HCl (pH=7.5) buffer to soak at twice 4～6 hours;
5) centrifugal treating, sediment separate out, with deposit by 1: 50 mass ratio distilled water diluting, dispersed with stirring, with about pure acetic acid regulation system pH value to 3.0, adding simultaneously accounts for 3% pepsin of deposit quality, 10 ℃ of stirring reactions 24 hours;
6) add EDTA in the reaction system, make its concentration reach 1000ppm, continue to stir 0.5 hour;
7) the reactant liquor frozen centrifugation is handled, and removes deposit, and the saturated sodium hydroxide solution of adding is neutralized to neutrality in the clear liquid;
8) and then add sodium chloride, reach 2mol/l until its concentration; Slowly stir, leave standstill then and saltout 2～12 hours;
9) frozen centrifugation collecting precipitation to distill water dialysis 48 hours, changed one time water every 4 hours;
10) after frozen centrifugation was handled, the precipitate lyophilization became collagen sponge, about 80g;
11) get in the distilled water that the 0.5g collagen sponge is dissolved in pH=3.0, add the 0.1g chitose, stirred crosslinked 1 hour under the room temperature as cross-linking agent;
12) product lyophilization once more, 100～120 ℃ of heat treated further crosslinked and sterilization in 1 hour under vacuum condition then;
13) under the aseptic condition product is added the sterile phosphate buffer solution of 20ml pH=6.8, make its abundant water absorption and swelling, knead into paste, add the 0.5g heparin then, 0.1g hyaluronic acid, 0.3% lignocaine, fully mix, under the aseptic condition paste is sealed in the disposable syringe, every pipe encapsulation 1～2ml preserves below 10 ℃.
Embodiment three:
The method for preparing packing material of the present invention is as follows:
1) preparation method of collagen is described with embodiment one or embodiment two;
2) get in the distilled water that the 0.5g collagen sponge is dissolved in pH=3.0, add the 0.1g chitose, stir crosslinked 1h under the room temperature as cross-linking agent;
3) product lyophilization once more, 100～120 ℃ of heat treated further crosslinked and sterilization in 1 hour under vacuum condition then;
4) under the aseptic condition product is added the sterile phosphate buffer solution of 20ml pH=6.8, make its abundant water absorption and swelling, knead into paste, add 0.3 heparin then, the 0.1g dermatan sulfate, cytokine bFGF lyophilized powder 10 μ g, 0.3% lignocaine fully mixes, and under the aseptic condition paste is sealed in the disposable syringe, every pipe encapsulation 1～2ml preserves below 10 ℃.
Claims (10)
1, a kind of injectable type collagen base soft tissue filling material is characterized in that mainly being made of following component: soluble collagen 0.5～20%; Heparin 0.1～10%; Cell growth factor 0～1%; PH value is 6.8～7.2 buffer 70～99.4%.
2, packing material according to claim 1 is characterized in that mainly being made of following component:
Soluble collagen 2～10%; Heparin 0.5～5%; Cell growth factor 0.0001%～0.001%; The phosphate buffer 85～97.1% of pH7.2; Local anesthetic 0.3%.
3, packing material according to claim 2 is characterized in that: described cell growth factor is bFGF, and described local anesthetic is a lignocaine.
4, according to the preparation method of claim 1,2 or 3 described packing materials, it is characterized in that: from animal connective tissue, extract soluble collagen, utilize the antigen decision family in the protease hydrolysis excision tropocollagen molecule, after the purification process, adopt the cross-linking agent of safety non-toxic crosslinked, add biological active component then, mixture is kneaded into paste repeatedly, sealed under aseptic conditions and low temperature storage.
5, preparation method according to claim 4 is characterized in that: described animal connective tissue comprises skin, cartilage or the tendon tissue that derives from pig, cattle or sheep.
6, preparation method according to claim 4 is characterized in that: described protease is pepsin, trypsin or bromelain.
7, preparation method according to claim 4 is characterized in that: described purification process is to remove wherein inorganic salt and small molecular weight impurity by dialysis or ultrafiltration membrane treatment.
8, preparation method according to claim 4 is characterized in that: described cross-linking agent is carbodiimides or chitose; When adopting carbodiimides, the final concentration of carbodiimides is 0.5～5%; When adopting chitose, the final concentration of chitose is 1%～20%.
9, preparation method according to claim 4 is characterized in that: described biological active component is one or more in cytokine composition, heparin and mucopolysaccharide, chondroitin sulfate, dermatan sulfate, hyaluronic acid or the chitose.
10, preparation method according to claim 4, it is characterized in that: described crosslinked be after with the soluble collagen lyophilization, add cross-linking agent, after carrying out 0.1～4 hour chemical crosslink reaction, handled 1～48 hour-80～0 ℃ of following lyophilization again, under vacuum condition, be heated to 100～120 ℃ then and handle 0～72 hour further heat cross-linking and sterilization.
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| CNB2004100278973A CN1294994C (en) | 2004-07-05 | 2004-07-05 | Injectable type collagen-based soft tissue filling material and preparation method thereof |
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| CNB2004100278973A CN1294994C (en) | 2004-07-05 | 2004-07-05 | Injectable type collagen-based soft tissue filling material and preparation method thereof |
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| CN1294994C true CN1294994C (en) | 2007-01-17 |
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Families Citing this family (13)
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|---|---|---|---|---|
| CN101874903B (en) * | 2009-11-20 | 2013-04-10 | 胡庆柳 | Method for preparing collagen artificial skin |
| CN101845226B (en) * | 2010-04-15 | 2011-12-07 | 四川大学 | Dialdehyde carboxymethyl cellulose-collagen frozen gel and preparation method thereof |
| CN101934092A (en) * | 2010-08-27 | 2011-01-05 | 中国人民解放军第三军医大学第一附属医院 | Injection-type cartilage bionic matrix for regenerative repair of cartilage and method for using same |
| AU2011334599B2 (en) * | 2010-11-23 | 2016-12-01 | Allergan Pharmaceuticals International Limited | Preparation and/or formulation of proteins cross-linked with polysaccharides |
| AU2012204311B2 (en) * | 2011-01-06 | 2017-05-04 | C. Lowell Parsons | Method for manufacturing composition comprising local anesthetic, heparinoid, and buffer |
| CN102488925A (en) * | 2011-12-29 | 2012-06-13 | 成都普川生物医用材料股份有限公司 | Injectable articular cartilage tissue repair material and its preparation method |
| CN104189956B (en) * | 2014-08-27 | 2016-03-16 | 陕西瑞盛生物科技有限公司 | A kind of injectable fills implant and preparation method thereof |
| CN105233270A (en) * | 2015-11-12 | 2016-01-13 | 上海昊海生物科技股份有限公司 | Medicine composition for water-optical treatment and application of medicine composition |
| CN106474077A (en) * | 2015-11-30 | 2017-03-08 | 湖南恒生制药股份有限公司 | The preparation method of injection chondroitin sulfate |
| CN107308494A (en) * | 2017-07-27 | 2017-11-03 | 北京华信佳音医疗科技发展有限责任公司 | A kind of injection collagen, preparation method and filler |
| CN107812244B (en) * | 2017-10-25 | 2020-09-29 | 北京华信佳音医疗科技发展有限责任公司 | Preparation of liquid collagen filler |
| CN108939153A (en) * | 2018-08-17 | 2018-12-07 | 振德医疗用品股份有限公司 | A kind of collagen-based packing material and preparation method thereof and its application method |
| US20230201418A1 (en) * | 2021-12-28 | 2023-06-29 | Shanghai Qisheng Biological Preparation Co., Ltd. | Soft tissue augmentation using injectable, neutral ph soluble collagen-glycosaminoglycan compositions |
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| CN1377257A (en) * | 1999-10-05 | 2002-10-30 | 核转移治疗公司 | Hybrid matrices and hybrid matrix mixtures |
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2004
- 2004-07-05 CN CNB2004100278973A patent/CN1294994C/en not_active Expired - Fee Related
Patent Citations (4)
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| CN1133562A (en) * | 1993-09-07 | 1996-10-16 | 资料范围投资公司 | Injectable compositions for soft tissue augmentation |
| CN1370084A (en) * | 1999-08-13 | 2002-09-18 | 白奥佛姆公司 | Tissue augmentation material and methods |
| CN1377257A (en) * | 1999-10-05 | 2002-10-30 | 核转移治疗公司 | Hybrid matrices and hybrid matrix mixtures |
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