CN104225689A - Preparation method of fish collagen bone-induced regeneration membrane - Google Patents
Preparation method of fish collagen bone-induced regeneration membrane Download PDFInfo
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- CN104225689A CN104225689A CN201310227115.XA CN201310227115A CN104225689A CN 104225689 A CN104225689 A CN 104225689A CN 201310227115 A CN201310227115 A CN 201310227115A CN 104225689 A CN104225689 A CN 104225689A
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Abstract
The invention discloses a preparation method of a fish collagen bone-induced regeneration membrane; aiming at physiological and biochemical characteristics of fish collagen protein hydrogel, the method adopts freeze drying and mechanical loading to prepare a 'dense layer-porous layer' double-layer fish collagen bone-induced regeneration membrane material. The method is characterized in that with fish scales as raw material, the collagen protein is extracted through a flash extraction method; and the extracted fish collagen protein is utilized to prepare the hydrogel, and the double-layer bone-induced regeneration membrane is prepared through freeze drying and mechanical loading. In the whole preparation process, the collagen protein in the extraction stage has less loss and high purity, the method of the preparation stage is simple and easy to implement, and the prepared bone-induced regeneration membrane has a 'dense layer-porous layer' double-layer structure, and is good in mechanical strength, good in water-absorbing property, good in biocompatibility, and good in application prospect.
Description
Technical field
The present invention relates to a kind of technology of preparing of the research field for self-bone grafting regeneration membrane, specifically, take fish scale as raw material, extract fish collagen by homogenate extraction method, then by the method for Mechanical loading fish collagen made there is " compacted zone-porous layer " double-deck self-bone grafting regeneration membrane.
Background technology
Modern more and more payes attention to the health of tooth, and Alveolar Bone Defect is one of key factor threatening dental health.The method of effective alveolar bone defect repair adopts self-bone grafting regeneration techniques exactly at present, and the key of self-bone grafting regeneration techniques is exactly the selection of self-bone grafting regeneration membrane.The composition of self-bone grafting regeneration membrane, structure, the success of character on operation produce important impact.
At present, conventional self-bone grafting regeneration membrane has titanium film, polymeric membrane, collagem membrane etc.Wherein, collagem membrane has absorbable characteristic, degrades gradually in human body, takes out, and has good biocompatibility and cellular affinity, seldom cause inflammatory reaction without the need to second operation.Therefore, collagen is used to be the focus of current bio-medical material research as self-bone grafting regeneration membrane prepared by raw material.Collagem membrane most popular is in the market the Bio-gide collagem membrane that Geistlich company of Switzerland produces, and its raw materials is pig collagen.This collagem membrane price is very expensive, for patient causes very high financial burden; And collagen sources is Lu Sheng mammal, itself and human homology's property are higher, easily cause some diseases to propagate between people and animals, simultaneously due to the restriction of religion or culture, can not use at all regions.To sum up, be badly in need of at present developing the succedaneum of a kind of collagem membrane with low cost as existing collagem membrane, meanwhile, collagen can not come from Lu Sheng mammal.Therefore, fish collagen as the raw material preparing self-bone grafting regeneration membrane, is extracted highly purified fish collagen by homogenate extraction method by us from discarded fish scale, with low cost, has wide market prospect.
Summary of the invention
The object of the invention is the deficiency overcoming existing self-bone grafting regeneration membrane, adopt method with low cost, simple to operate to obtain collagen self-bone grafting regeneration membrane.This preparation method can meet the needs of low cost large-scale production collagen self-bone grafting regeneration membrane.
First the present invention extracts fish collagen by homogenate extraction method from discarded fish scale, then forms collagen gel by dialysis, prepares double-deck collagen self-bone grafting regeneration membrane finally by vacuum lyophilization and Mechanical loading mode.Comprise following processing step: 1. homogenate extraction method extracts fish collagen.(1) fish scale pretreatment: the fish skin of removing fish scale adhesion, cleans fish scale, dries.Pending fish scale is shredded with pulverizer; (2) distilled water is added, homogenate extraction 2 times; (3) collagen protein extracts: add low-concentration acetic acid solution with swelling ratio 1:15-30, mix homogeneously, places 4h, and afterwards with the centrifugal 5-30min of 3500-5500r/min rotating speed, removing impurity, gets supernatant, for subsequent use; (4) except foreigh protein removing: crude extract and NaCl solution are mixed by weight ratio, with the centrifugal 5-30min of 3500-5500r/min rotating speed, abandoning supernatant, precipitates the acetic acid with the 0.5mol/L of 10 times of volumes, with the centrifugal 5-30min of 3500-5500r/min rotating speed.Repeat this step about 3 times; 2. dialyse: desalination and acid, form gel simultaneously.Dissolved by the precipitation 10-50 obtained after saltouing several times distilled water doubly, dialysis 3-7 days, changed first water every several hours, after 3-7 days, collagen forms the gel being applicable to the optimum state preparing self-bone grafting regeneration membrane, and wherein bag filter aperture is 7000.3. vacuum lyophilization sample: by dialysis after collagen gel be positioned in flat-type container, carry out drying with vacuum freeze drier, period take certain hour as the cycle, carries out freezing melt process to sample, collagen protein sample.4. Mechanical loading: by drying completely fish collagen sample be placed in mechanics apparatus pressure apparatus, with the speed range of the scope of 0-8000N and 0.1mm/s-0.5mm/s, Collagen specimens is carried out to the loading of pressure, namely can obtain the double-deck self-bone grafting regeneration membrane prepared.
In the present invention, first, when extracting fish collagen, fish scale pretreatment, acid are carried, deacidify and the technological temperature of salt is room temperature, except foreigh protein removing temperature is 4 DEG C.The concentration of low-concentration acetic acid solution is 0.1-1mol/L, and it can remove fribrillin, and fish skin is softened, and collagen protein comparatively foreign protein is stablized.The acetum of low concentration can't cause collagen to degrade in a large number when being hydrolyzed and removing foreign protein.Low-concentration acetic acid is collaborative to be extracted, and the collagen protein obtained can keep its triple helix structure to greatest extent, is applicable to biomedical material and raw material thereof.The concentration of NaCl solution is 0.1-1mol/L, and certain density NaCl can make the foreign protein in fish skin separate out, and assists in removing foreign protein Purified collagen.Secondly, when dialysing to collagen solution, selection natural law is 3-7 days, and the time of the dialysis of selection is conducive to the thorough removing of acid and salt, is also conducive to the gel forming the most applicable follow-up preparation process.In addition, when carrying out vacuum lyophilization, selection cycle carries out melt process to collagen, by the degree of cross linking improving collagen-based materials that colds and heat succeed each other, can improve the mechanical strength of final self-bone grafting regeneration membrane; Meanwhile, material remains on invariant position on plate container, by the wherein evaporation of moisture and the distillation of ice crystal, naturally form " compacted zone-porous layer " double-deck material.Finally, select the pressure of 0-8000N, in this pressure limit, enough power can be provided, change volume and the thickness of fish collagen material, become the form being suitable for the self-bone grafting regeneration membrane for the treatment of most.
Compared with prior art, the present invention adopts fish scale to be raw material, effectively reduces the cost of material of self-bone grafting regeneration membrane; Adopt homogenate extraction method, extraction rate is fast, and cost is low, reduces the extraction cost of collagen; Take Mechanical loading to prepare the self-bone grafting regeneration membrane of film shape, method is simple, with short production cycle, and output is high, and easily realize industrialization.Obtained collagen self-bone grafting regeneration membrane mechanical strength is good, biocompatibility is good, double-decker plays the effect of barrier and guiding simultaneously: " compacted zone " isolates non-osteoblast, for Oesteoblast growth provides space, " porous layer " provides growth attachment point for osteoblast, and therefore this materials application scope is wide.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of the regeneration induction film adopting the inventive method to prepare
Fig. 2 is the pictorial diagram of the regeneration induction film adopting the inventive method to prepare
Fig. 3 is the scanning electron microscope (SEM) photograph of the regeneration induction film adopting the inventive method to prepare
Fig. 4 is the infared spectrum of the regeneration induction film adopting the inventive method to prepare
Detailed description of the invention
The present invention is further elaborated by following instance, but does not limit the scope of the invention.The production technology of this manufacturing technology for this specialty personnel be easily implement.
It is raw material that example 1 chooses tilapia fish scale, and other materials of fish skin and adhesion on removing fish scale, clean and shred stand-by.By distilled water with fish scale by weight ratio for 15-30:1 mixes, dodge carry twice, each 1.5 minutes.Be the acetic acid solution that 1:15-30 adds 0.5mol/L by swelling ratio, afterwards with the centrifugal 5-30min of 3500-5500r/min rotating speed, get supernatant, for subsequent use.Crude extract and NaCl solution are mixed by weight ratio, with the centrifugal 5-30min of 3500-5500r/min rotating speed, abandoning supernatant, precipitates the acetic acid with the 0.5mol/L of 10 times of volumes, with the centrifugal 5-30min of 3500-5500r/min rotating speed.Repeat this step about 3 times.Dialysed three days by the supernatant obtained after saltouing several times, per half a day changes first water.Bag filter aperture is 7000.By dialysis after collagen solution vacuum freeze drier carry out drying, collagen protein semifinished product.
By dialysis after collagen gel be positioned in flat-type container, carry out drying with vacuum freeze drier, period take certain hour as the cycle, carries out freezing melt process to sample, collagen protein sample.Again by drying completely fish collagen sample be placed in mechanics apparatus pressure apparatus, with the speed range of the scope of 0-8000N and 0.1mm/s-0.5mm/s, Collagen specimens is carried out to the loading of pressure, namely can obtain the double-deck self-bone grafting regeneration membrane prepared.
It is raw material that example 2 chooses Cyprinus carpio fish scale, and other materials of fish skin and adhesion on removing fish scale, clean and shred stand-by.By distilled water with fish scale by weight ratio for 15-30:1 mixes, dodge carry twice, each 1.5 minutes.Be the acetic acid solution that 1:15-30 adds 0.5mol/L by swelling ratio, afterwards with the centrifugal 5-30min of 3500-5500r/min rotating speed, get supernatant, for subsequent use.Crude extract and NaCl solution are mixed by weight ratio, with the centrifugal 5-30min of 3500-5500r/min rotating speed, abandoning supernatant, precipitates the acetic acid with the 0.5mol/L of 10 times of volumes, with the centrifugal 5-30min of 3500-5500r/min rotating speed.Repeat this step about 3 times.Dialysed three days by the supernatant obtained after saltouing several times, per half a day changes first water.Bag filter aperture is 7000.
By dialysis after collagen gel be positioned in flat-type container, carry out drying with vacuum freeze drier, period take certain hour as the cycle, carries out freezing melt process to sample, collagen protein sample.Again by drying completely fish collagen sample be placed in mechanics apparatus pressure apparatus, with the speed range of the scope of 0-8000N and 0.1mm/s-0.5mm/s, Collagen specimens is carried out to the loading of pressure, namely can obtain the double-deck self-bone grafting regeneration membrane prepared.
It is raw material that example 3 chooses grass carp scales, and other materials of fish skin and adhesion on removing fish scale, clean and shred stand-by.By distilled water with fish scale by weight ratio for 15-30:1 mixes, dodge carry twice, each 1.5 minutes.Be the acetic acid solution that 1:15-30 adds 0.5mol/L by swelling ratio, afterwards with the centrifugal 5-30min of 3500-5500r/min rotating speed, get supernatant, for subsequent use.Crude extract and NaCl solution are mixed by weight ratio, with the centrifugal 5-30min of 3500-5500r/min rotating speed, abandoning supernatant, precipitates the acetic acid with the 0.5mol/L of 10 times of volumes, with the centrifugal 5-30min of 3500-5500r/min rotating speed.Repeat this step about 3 times.Dialysed three days by the supernatant obtained after saltouing several times, per half a day changes first water.Bag filter aperture is 7000.
By dialysis after collagen gel be positioned in flat-type container, carry out drying with vacuum freeze drier, period take certain hour as the cycle, carries out freezing melt process to sample, collagen protein sample.Again by drying completely fish collagen sample be placed in mechanics apparatus pressure apparatus, with the speed range of the scope of 0-8000N and 0.1mm/s-0.5mm/s, Collagen specimens is carried out to the loading of pressure, namely can obtain the double-deck self-bone grafting regeneration membrane prepared.
Claims (8)
1. prepare a method for collagen self-bone grafting regeneration membrane, it is characterized in that: take fish scale as raw material, adopt homogenate extraction method to extract fish collagen; Adopt lyophilization, Mechanical loading is prepared has " compacted zone-porous layer " double-deck self-bone grafting regeneration membrane.Self-bone grafting regeneration membrane preparation method of the present invention, can be prepared into collagen self-bone grafting regeneration membrane by the fish collagen extracted in fish scale easily.
2. preparation method according to claim 1, is characterized in that, described fish scale raw material, specifically derives from Cyprinus carpio, Carassius auratus, Mylopharyngodon piceus, Ctenopharyngodon idellus, Hypophthalmichthys molitrix, Aristichthys nobilis, tilapia etc.; The self-bone grafting regeneration membrane prepared has thin and " compacted zone " of densification and thicker and loose porous " porous layer ".
3. a kind of method preparing collagen self-bone grafting regeneration membrane according to claim 1, it is characterized in that, described method is a kind of brand-new preparation method, adopt homogenate extraction method to prepare fish collagen fast, employing operational approach preparation method that is simple, applied range prepares double-deck collagen self-bone grafting regeneration membrane.It can meet the needs that collagen self-bone grafting regeneration membrane is produced.
4. prepare a method for fish collagen fast, it is characterized in that comprising following steps: comprise following processing step:
(1). homogenate extraction method extracts fish collagen.
A) fish scale pretreatment: the fish skin of removing fish scale adhesion, cleans fish scale, dries.Pending fish scale is shredded with pulverizer;
B) distilled water is added, homogenate extraction 2 times;
C) collagen protein extracts: add low-concentration acetic acid solution with swelling ratio 1:15-30, mix homogeneously, places 4h, and afterwards with the centrifugal 5-30min of 3500-5500r/min rotating speed, removing impurity, gets supernatant, for subsequent use;
D) except foreigh protein removing: crude extract and NaCl solution are mixed by weight ratio, with the centrifugal 5-30min of 3500-5500r/min rotating speed, abandoning supernatant, precipitates the acetic acid with the 0.5mol/L of 10 times of volumes, with the centrifugal 5-30min of 3500-5500r/min rotating speed.Repeat this step about 3 times;
(2). dialysis: desalination and acid, form gel simultaneously.
Dissolved by the precipitation 10-50 obtained after saltouing several times distilled water doubly, dialysis 3-7 days, changed first water every several hours, after 3-7 days, collagen forms the gel being applicable to the optimum state preparing self-bone grafting regeneration membrane, and wherein bag filter aperture is 7000.
(3). vacuum lyophilization sample.
By dialysis after collagen gel be positioned in flat-type container, carry out drying with vacuum freeze drier, period take certain hour as the cycle, carries out freezing melt process to sample, collagen protein sample.
(4). Mechanical loading.
By drying completely fish collagen sample be placed in mechanics apparatus pressure apparatus, with the speed range of the scope of 0-8000N and 0.1mm/s-0.5mm/s, Collagen specimens is carried out to the loading of pressure, namely can obtain the double-deck self-bone grafting regeneration membrane prepared.
5. preparation process according to claim 4, its temperature profile is, described fish scale pretreatment, acid are carried, deacidify and the technological temperature of salt is room temperature, except foreigh protein removing temperature is 4 DEG C.Acetum is characterised in that, described low-concentration acetic acid solution, and its concentration is about 0.1-1mol/L.Acetic acid can remove fribrillin, and fish skin is softened, and collagen protein comparatively foreign protein is stablized, and the acetum of low concentration can't cause collagen to degrade in a large number when being hydrolyzed and removing foreign protein.Low-concentration acetic acid is collaborative to be extracted, and the collagen protein obtained can keep its triple helix structure to greatest extent, is applicable to biomedical material and raw material thereof.Saline solution, is characterized in that, described saline solution refers to NaCl solution, and its concentration is 0.1-1mol/L, and certain density NaCl can make the foreign protein in fish skin separate out, and assists in removing foreign protein Purified collagen.
6. preparation process according to claim 4, its dialysis time is characterised in that, according to the difference of fish collagen swelling ratio, dialysis natural law 3-7 days is not etc., dialysis procedure can change ion concentration and the pH value of fish collagen solution, when ion concentration and pH value reach a particular range, collagen solution forms stable gel.
7. preparation process according to claim 4, its freezing melting characteristic is, the freezing melting of different number of times can change the degree of cross linking of collagen gel, thus changes the mechanical property of collagen self-bone grafting regeneration membrane.
8. preparation process according to claim 4, its Mechanical loading scoped features is 0-8000N.Mechanical loading can change internal structure and the physical characteristic of collagen protein block, under the effect of this loading force, can not destroy the structure of fish collagen, obtains the self-bone grafting regeneration membrane of suitable thickness simultaneously.
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| CN105601666A (en) * | 2016-01-12 | 2016-05-25 | 武汉轻工大学 | Method for extracting phospholipid from heads of hypophthalmichthys molitrix and product of method |
| CN109432500A (en) * | 2018-12-04 | 2019-03-08 | 冠昊生物科技股份有限公司 | A kind of preparation method and application of collagen membrane support |
| CN109939256A (en) * | 2019-04-01 | 2019-06-28 | 南京华开生物科技有限公司 | A kind of asymmetric dressing for skin and preparation method thereof |
| CN111303454A (en) * | 2020-03-10 | 2020-06-19 | 广西医科大学 | Preparation method and application of crocodile skin collagen hydrogel |
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| CN111303454A (en) * | 2020-03-10 | 2020-06-19 | 广西医科大学 | Preparation method and application of crocodile skin collagen hydrogel |
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Application publication date: 20141224 |