CN1319604C - Double layer composite collagen base guide tissue regeneration film and its preparing method - Google Patents
Double layer composite collagen base guide tissue regeneration film and its preparing method Download PDFInfo
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- CN1319604C CN1319604C CNB2004100719321A CN200410071932A CN1319604C CN 1319604 C CN1319604 C CN 1319604C CN B2004100719321 A CNB2004100719321 A CN B2004100719321A CN 200410071932 A CN200410071932 A CN 200410071932A CN 1319604 C CN1319604 C CN 1319604C
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Abstract
The present invention relates to a regenerating material of a double-layer compound collagen-base guiding tissue and a preparing method thereof. Collagen and hyaluronic acid or the sodium salt of the hyaluronic acid are used as main raw materials for preparing a double-layer compound material with the structure of a compact layer and an areosis layer. The mass composition is as following: the compact layer is composed of 0.6% to 0.8% of malonic acid collagen swelling liquid, and the areosis layer is composed of 0.2% to 0.4% of malonic acid collagen swelling liquid and hyaluronic acid of which the collagen dry weight content is from 5% to 20% or the sodium salt of the hyaluronic acid. The present invention has good biocompatibility, one side is an areosis layer, and the other side is a compact structure; thus, the product has the obvious characteristics of preventing mechanocytes, etc., from growing into a coloboma part, and promoting the regeneration and the repair of the tissue of the coloboma part, and the characteristics of corresponding biological functions and degradable absorption. The implantation material is not required to be taken out by a secondary operation. The present invention is suitable for the tissue coloboma caused by wounds, tumor operations, etc., and is used for the guiding regeneration and repair of the coloboma tissue to realize the regenerating cure of the coloboma tissue.
Description
Technical field
The invention belongs to biomedical materials field, particularly relate to a kind of double layer composite collagen base guide tissue regeneration material and preparation method thereof.The present invention is applicable to the tissue defect that causes owing to wound, tumor post-operation etc., is used for the guiding Regeneration and Repair of defective tissue, realizes the healing by regeneration of defective tissue.As a kind of biomaterial that function is arranged, the prepared material of the present invention also can be used as the research and development that tissue engineering bracket is used for engineered artificial organ.
Technical background
Medical guide tissue regeneration material is the important component part of biomaterial for medical purpose, is the brand-new biomaterial of a class that developed in recent years.Guide tissue regeneration (guided tissue regeneration, GTR) notion eighties is at first by propositions such as N yman, it is meant effects such as relying on mechanical barrier, optionally cell guiding to impaired position adhere to, hypertrophy, reach the purpose of tissue repair.Realize that the reparation of tissue sick (lacking) damage and the key of guided tissue regeneration are the development and application of functional repair materials.
As a kind of brand-new treatment means that promotes the tissue regeneration healing, guide tissue regeneration utilizes the protection of membrane material, iris action to realize tissue regeneration or reconstruction on form and the function cleverly, and the limitation of traditional operation therapy has been broken in the rise of this technology.This key problem in technology is the application of guide tissue regeneration film.Existing guiding regrown material great majority are by the homogenous material preparation, are divided into degradable and non-degradable two big classes.The seventies later stage is to the eighties initial stage, people such as Nyman, Gottlow, Becker study non-degradable material polyacetals, polytetrafluoroethylene (PTFE), silicone coated etc. in succession, though this class material has better biocompatibility with tissue, but because of not being organized absorption, need second operation to take out material, increased the chance of wound.
Traditional guide tissue regeneration mainly is the barrier film effect that utilizes some biomaterial to form, make the damaged tissues part avoid the influence of fibrous connective tissue etc. on every side, thereby help regeneration and restoration, but do not have good facilitation for the tissue that needs reparative regeneration.For more helping tissue repair regeneration, need the material of development of new, the novel GTR material of being developed it should be able to stop fibroblast etc. to the growing into of damaged place, and also should promote the Regeneration and Repair of damaged place tissue to have biological function.Have the guide tissue regeneration material of multiple structure can realize this purpose by preparation, more existing research work make progress.In these research work, (as the material of selecting the preparation of two Collagen Type VIs for use makes its main cartilaginous tissue that is applicable to) or preparation technology's unstability (as prepare the separation that hierarchy may appear in multi-layer film material by mechanical compaction) are affected these goods practicality that are developed into because the restriction of main material.Selecting widely used type i collagen for use is main material, utilize the biological function of promotion cell adhesion, propagation and differentiation that hyaluronic acid or its sodium salt have simultaneously, adopt more stable and easy preparation technology, can better realize the clinical needs that guide tissue regeneration is repaired.
Summary of the invention
The purpose of this invention is to provide a kind of double layer composite collagen base guide tissue regeneration material and preparation method thereof, it is to be the medical guide tissue regeneration material of primary raw material preparation with the collagen protein that extracts from beef tendon.The new medical guide tissue regeneration film that the present invention makes, the characteristics that have collagen-based materials and hyaluronic acid or its sodium salt material simultaneously, biocompatibility is good, and prepared material is the sided configuration difference, and a side is an open structure, opposite side is a compact texture, goods are had to stop fibroblast etc. to the growing into of damaged place, promote the Regeneration and Repair of damaged place tissue to have corresponding biological function simultaneously, degradable absorbs, the distinguishing feature of needn't second operation taking out embedded material.
The present invention includes the collagen protein that from the connective tissue of animal, extracts with the protease digestion method, malonic acid solution-treated with 0.3% makes the collagen protein swelling, reuse concentration is 0.3% malonic acid solution adjusting solids content behind the mixing, promptly obtains collagen solution.Prepare the collagen solution of different content ratio respectively, wherein mixed transparent matter acid or its sodium salt in the collagen solution of low content ratio, the consumption of hyaluronic acid or its sodium salt is the 5-20% of collagen dry weight in the collagen solution of corresponding low content ratio, adopt the method molding of common lyophilization and extrusion forming, make collagen protein and hyaluronic acid or its sodium salt intermolecular cross-linking after the film forming, last after drying or the crosslinked composite collagen membrane material of lyophilizing with conventional cross-linking method.
For realizing purpose of the present invention, at first use mammiferous connective tissue such as beef tendon, Corii Bovis seu Bubali, pig tendon, Corii Sus domestica etc. and carry out the enzyme processing, prepare collagen solution and see Chinese patent ZL94118836.1.Particular content comprises that the employing enzyme digestion extracts type i collagen from the beef tendon tissue, and removes having antigenic end peptide in the collagen fiber, makes it have excellent biological compatibility.Select medical grade hyaluronic acid or its sodium salt for use and, make it have good composite performance through processing such as peroxidating.By blend, repeatedly, multistage freeze drying technology and chemical crosslinking, prepare double-deck composite, a side is to contain the hyaluronic acid of collagen and appropriate ratio or the weaker zone of its sodium salt in the material, opposite side is the compacted zone that contains high concentration ratio collagen-based materials.Be specially and take by weighing lyophilizing processing under an amount of collagen swelling solution appropriateness dilution back precooling, the appropriate condition in proportion, the freeze-dried material post processing becomes compact texture; Taking by weighing collagen swelling solution and hyaluronic acid or its sodium salt respectively in suitable ratio dilutes respectively and dissolves, in homodisperse collagen diluent, slowly add hyaluronic acid or its sodium salt solution, limit edged stirring at low speed makes the two mixing, the pre-freeze appropriate time is formed with the frozen water structure of certain viscosity, after the collagen compacted zone that will prepare closely cover thereon, continue freezing and carry out lyophilizing and handle, become loose relatively/fine and close structure; Freeze dried composite cleans lyophilizing Processing of Preparation finished product through chemical crosslinking.The used cross-linking agent of chemical crosslinking can be selected cross-linking agent commonly used such as aldehydes, diimine class for use.The product that obtains is cut into required form, packs with aluminium foil or other flexible packing materials, uses the gamma-radiation radiation sterilization then.Can adjust the thickness that compacted zone and weaker zone thickness are adjusted final finished as required in the preparation process.
Specific embodiments of the present invention is: the double layer composite collagen base guide tissue regeneration material is to be that main raw material(s) is prepared into the double-layer composite material with compacted zone and weaker zone structure with collagen and hyaluronic acid or its sodium salt, and quality group becomes:
Described compacted zone is malonic acid collagen swelling solution 0.6-0.8%.
Described weaker zone is hyaluronic acid or its sodium salt of malonic acid collagen swelling solution 0.2-0.4% and collagen dry weight content 5-20%.
Described double layer composite collagen base guide tissue regeneration preparation methods comprises the steps:
(1) extracts preparation collagen swelling solution by method described in the patent 94118836.1, be mixed with malonic acid collagen swelling solution 0.6-0.8% by the malonic acid that measures with 0.3%;
(2) malonic acid collagen swelling solution is poured in the suitable containers into the refrigerator 2 hours that places-35 ℃, thickness 1-3cm, then in vacuum freeze drier, lyophilization;
(3) collagem membrane is placed between the flat board of two suitable sizes, the machinery pressurization makes into relative compacted zone;
Under (4) 4 ℃, medical grade hyaluronic acid or its sodium salt are carried out oxidation processes 18 hours with the oxidant sodium periodate solution, use the remaining oxidant of bisulfite sodium reduction unreacted then, till colourless;
Under (5) 4 ℃, in a large number to water dialysis 72 hours, ℃ pre-east then-30, lyophilizing is stand-by in the vacuum freeze drier.
(6) get homodisperse malonic acid collagen swelling solution 0.4-0.8% by metering and become solution with 0.3% malonic acid or water dissolution with hyaluronic acid or its sodium salt through above-mentioned processing, the 5-20% that hyaluronic acid or its sodium salt consumption are the collagen dry weight, add hyaluronic acid or its sodium salt solution in the collagen swelling solution, making collagen swelling solution final concentration is 0.2-0.4%, stirring and evenly mixing, be transferred in the suitable vessel thickness 1-3cm ,-30 ℃ of pre-freezes 30 minutes; Then the above-mentioned collagen compacted zone that has prepared is closely covered thereon ,-30 ℃ are continued freezing and carry out lyophilizing and handle;
(7) composite of above-mentioned molding is crosslinked with crosslinker solution commonly used, crosslinked back is extremely neutral with distilled water thorough washing and soaked overnight; And then be placed in the suitable vessel, and freeze-drying method lyophilizing;
(8) from freeze dryer, after the taking-up, be placed on the flat board of two suitable sizes, flatten, become end article;
(9) cut into different specifications, packing is shone sterilization with gamma-radiation then, promptly obtains the composite collagen base guide tissue regeneration material.
Described weaker zone is that hyaluronic acid or its sodium salt of 0.2-0.4% collagen swelling solution and collagen dry weight content 5-20% formed.
Hyaluronic acid or its sodium salt solution add in the collagen swelling solution in the described weaker zone preparation process, can also regulate pH value to 7-9 with the Tris alkali of 0.1M or the sodium bicarbonate solution of 0.1M, put stirring at low speed in 20 ℃ of Tempeerature-constant air bath cabinets,＜60 commentaries on classics/per minutes 24 hours, make the two that cross-linking reaction take place, after reaction finishes, carefully discard too much liquid, remaining solid suspension-30 ℃ pre-freeze is to forming weaker zone.
The new medical guide tissue regeneration film that the present invention makes, the characteristics that have collagen-based materials and hyaluronic acid or its sodium salt material simultaneously, biocompatibility is good, and prepared material is the sided configuration difference, and a side is an open structure, opposite side is a compact texture, goods are had to stop fibroblast etc. to the growing into of damaged place, promote the Regeneration and Repair of damaged place tissue to have corresponding biological function simultaneously, degradable absorbs, the distinguishing feature of needn't second operation taking out embedded material.The present invention is applicable to the tissue defect that causes owing to wound, tumor post-operation etc., is used for the guiding Regeneration and Repair of defective tissue, realizes the healing by regeneration of defective tissue.As a kind of biomaterial, the prepared material of the present invention also can be used as the research and development that tissue engineering bracket is used for engineered artificial organ.
Description of drawings
Fig. 1 duplicature dense side surface scan sem image (* 50).
Fig. 2 duplicature dense side surface scan sem image (* 50).
The specific embodiment
Adopt method described in the Chinese patent ZL94118836.1 (CN 1086145C) to extract preparation collagen swelling solution, swelling solution is diluted to 0.6% with 0.3% malonic acid, it is 100mm that swelling solution 100 grams of collagen protein dilution are poured diameter into, highly is in the glass dish of 2.5cm.Plate is placed-35 ℃ refrigerator 2 hours earlier, it is lyophilization 50 hours under the 10-100u that the plate that rapidly this is freezed after the taking-up places on the shelf of vacuum freeze drier in vacuum.From freeze dryer, take out freeze dried collagen-based materials and place between two politef flat boards, flatten, make into relative compacted zone with smooth object such as rollers.
Medical grade hyaluronate sodium 1 gram is dissolved in the 50ml water, with 20mM sodium periodate solution 50ml, 0.2M sodium perchlorate 5ml carries out oxidation processes, 4 ℃ 18 hours, reaction finishes the remaining oxidant of bisulfite sodium reduction unreacted that the back slowly drips 0.04M, determine addition according to change color, to color be colourless till, in a large number water is dialysed, 4 ℃ 72 hours, change water for dialysis every day 4 times, be transferred to-30 ℃ of pre-freezes in the suitable vessel, lyophilizing is stand-by in the vacuum freeze drier.
Take by weighing collagen swelling solution and treated hyaluronate sodium respectively in suitable ratio, dilute respectively and dissolve, making collagen diluted swelling liquid concentration is 0.6%50ml, 15% (mass ratio) of the relative collagen dry weight content of hyaluronate sodium consumption, with 0.3% malonic acid or water dissolution in becoming 50ml solution, slowly add sodium hyaluronate solution in the homodisperse collagen diluent, limit edged stirring at low speed makes the two mixing,-30 ℃ of pre-freeze appropriate times (about 30 minutes), be formed with the frozen water structure of certain viscosity, the collagen compacted zone that has prepared is closely covered thereon, continue freezing and carry out lyophilizing and handle, become the different composite of bilateral compactness extent.
With the composite concentration of the molding that makes as stated above is that pH is crosslinked 120 minutes of 8.4 formalin, crosslinked back with distilled water thorough washing 10-15 time and soaked overnight to neutrality.And then be placed in the glass dish lyophilizing of pre-freeze postlyophilization method.After from freeze dryer, taking out, once more film is placed two polyfluortetraethylene plates to flatten, become end article.
Cutting into different specifications, pack with aluminium foil or other flexible packing materials, is the gamma-radiation photograph sterilization of 25kGy with dosage then, promptly obtains the composite collagen base guide tissue regeneration material.Fig. 1 duplicature dense side surface scan sem image (* 50).Fig. 2 duplicature dense side surface scan sem image (* 50).
Embodiment 2
Fine and close collagen layer and hyaluronate sodium are handled with embodiment 1.
The weaker zone preparation process is: take by weighing an amount of collagen swelling solution and treated hyaluronate sodium, dilute respectively and dissolve, concentration and ratio are with embodiment 1, sodium hyaluronate solution is added in the collagen swelling solution, regulate pH value to 7-9 with the Tris alkali of 0.1M or the sodium bicarbonate solution of 0.1M, put the interior low speed (＜60 rpms) of 20 ℃ of Tempeerature-constant air bath cabinets and shook 24 hours, make hyaluronic acid sodium molecule and collagen generation cross-linking reaction through oxidation processes.After reaction finishes, carefully discard too much liquid, remaining solid suspension press condition pre-freeze among the embodiment 1, and the compacted zone that tight covering has prepared during to the frozen water structure that forms certain viscosity continues pre-freeze to becoming the ice sample structure fully, the lyophilizing processing.Lyophilizing sample press forming in machinery, section is packed, and the gamma-radiation that with dosage is 25kGy promptly obtains the composite collagen base guide tissue regeneration material according to sterilization.
Claims (3)
1, a kind of double layer composite collagen base guide tissue regeneration material, it is made up of the collagen layer and the weaker zone of compact texture, and the collagen layer that it is characterized in that described compact texture is to be formed by 0.6-0.8% malonic acid collagen swelling solution lyophilizing compacting; Described weaker zone is that the malonic acid or the aqueous solution lyophilizing of malonic acid collagen swelling solution and hyaluronic acid or its sodium salt forms, hyaluronic acid or its sodium salt consumption 5-20% that is the collagen dry weight wherein, the concentration of collagen in final mixed solution is 0.2-0.4%; The collagen compacted zone closely is overlying on repressed again molding after the continuation lyophilization on the weaker zone.
2, the said double layer composite collagen base guide tissue regeneration preparation methods of claim 1 is characterized in that it comprises the steps:
(1) method described in the Chinese patent ZL94118836.1 of pressing is extracted preparation collagen swelling solution, is mixed with malonic acid collagen swelling solution 0.6-0.8% by the malonic acid that measures with 0.3%;
(2) malonic acid collagen swelling solution is poured in the container into the refrigerator 2 hours that places-35 ℃, then in vacuum freeze drier, lyophilization;
(3) collagem membrane is placed two be fit to flatten between the big or small flat board, make into relative compacted zone;
Under (4) 4 ℃, hyaluronic acid and sodium salt thereof are carried out oxidation processes 18 hours with the oxidant sodium periodate solution, use the remaining oxidant of bisulfite sodium reduction unreacted then, till colourless;
Under (5) 4 ℃, in a large number to water dialysis 72 hours, ℃ pre-freeze then-30, lyophilizing is stand-by in the vacuum freeze drier;
(6) get homodisperse malonic acid collagen swelling solution by metering and through hyaluronic acid or its sodium salt of above-mentioned processing, this kind hyaluronic acid or its sodium salt are become solution with 0.3% malonic acid or water dissolution, add hyaluronic acid or its sodium salt solution in the collagen swelling solution, stirring and evenly mixing,-30 ℃ of pre-freezes 30 minutes, then the above-mentioned collagen compacted zone that has prepared is closely covered thereon ,-30 ℃ are continued freezing and carry out lyophilizing and handle;
(8) composite of above-mentioned molding is crosslinked with cross-linking agent solution commonly used, crosslinked back uses distilled water thorough washing 10-15 time and soaked overnight to neutral; And then be placed in the container freeze-drying method lyophilizing;
(9) from freeze dryer, take out after, film is placed between two flat boards, machinery adds and flattens, and becomes end article;
(10) cut into different specifications, packing is shone sterilization with gamma-radiation then, promptly obtains the composite collagen base guide tissue regeneration material.
3, double layer composite collagen base guide tissue regeneration preparation methods according to claim 2, it is characterized in that in the described weaker zone preparation process that hyaluronic acid or its sodium salt solution add in the collagen swelling solution, be to regulate pH value to 7-9 with the Tris alkali of 0.1M or the sodium bicarbonate solution of 0.1M, put stirring at low speed in 20 ℃ of Tempeerature-constant air bath cabinets,＜60 commentaries on classics/per minutes, 24 hours, make the generation cross-linking reaction, after reaction finishes, discard too much liquid, remaining solid suspension-30 ℃ pre-freeze is to forming weaker zone.
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| CN101954126A (en) * | 2010-09-26 | 2011-01-26 | 华南理工大学 | Method for preparing bionic modified collagen tissue repair material |
| CN102716517B (en) * | 2011-03-30 | 2015-01-14 | 深圳兰度生物材料有限公司 | Guided tissue regeneration membrane and its preparation method |
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| CN107551324B (en) * | 2017-10-27 | 2020-02-18 | 北京华信佳音医疗科技发展有限责任公司 | Preparation and application of suturable hard membrane repairing material |
| CN108744064B (en) * | 2018-06-27 | 2021-04-20 | 山东大学齐鲁医院 | Three-dimensional biological scaffold material and preparation method thereof |
| CN111035812A (en) * | 2019-12-20 | 2020-04-21 | 厦门大学附属中山医院 | Human-derived cell biological composite patch |
| CN113372436A (en) * | 2021-07-08 | 2021-09-10 | 中国海洋大学 | Medical collagen membrane with compact fish skin outer layer and loose inner layer and preparation method thereof |
| CN116570770B (en) * | 2023-07-12 | 2023-11-07 | 天新福（北京）医疗器材股份有限公司 | Double-layer freeze-drying tube and preparation method and application thereof |
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