CN104941008A - Preparation method and application of guided tissue regeneration membrane - Google Patents
Preparation method and application of guided tissue regeneration membrane Download PDFInfo
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- CN104941008A CN104941008A CN201510256512.9A CN201510256512A CN104941008A CN 104941008 A CN104941008 A CN 104941008A CN 201510256512 A CN201510256512 A CN 201510256512A CN 104941008 A CN104941008 A CN 104941008A
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Abstract
The invention discloses a preparation method and application of a guided tissue regeneration membrane. The method disclosed by the invention comprises the following steps: (1) soaking skin of an animal in vitro with a surfactant solution; (2) soaking a product in the step (1) with an alkali solution; (3) soaking the product in the step (2) with a peroxide solution; (4) soaking the product in the step (3) with an irradiating protection reagent solution; (5) soaking the product in the step (4) with a buffer liquid; and (6) sequentially carrying out freeze drying and irradiation sterilization on the product in the step (5), so as to obtain the guided tissue regeneration membrane. An experiment proves that the guided tissue regeneration membrane disclosed by the invention can be used for assisting osteanagenesis and guiding tissue growth.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of preparation method and application thereof of guide tissue regeneration film.
Background technology
The concept of guide tissue regeneration (GTR) was proposed by Nyman etc. first in nineteen eighty-two, just caused the concern of numerous scholars subsequently, during the reparation being applied to multiple hard and soft tissue and regeneration are studied, as Cranial defect, skin trauma and tendon rupture etc.The ultimate principle of guide tissue regeneration technology is the physical barriers function by film, by defect and surrounding tissue isolated and support certain organization survival space, prevention connective tissue, epithelial cell enter defective region, thus tissue repair regeneration capacity are farthest played.
It is conventional in Dental implantion that can film can be absorbed in vivo according to it, degrading is divided into nonabsorable film and absorbability film.The absorbability film with biodegradability is the more class material of current GTR technical research, must grasp in the use of this kind of material tissue regeneration and material degradation absorb between relation.Desirable absorbability film, can optionally guide tissue regeneration, and when this is done, film material will be completely degraded absorption.Therefore, absorbability GTRM should possess following condition: 1. optionally can guide cell regeneration; 2. there is good biological tissue's compatibility and cellular affinity; 3. there is good mechanical barrier effect, separate different cell; 4. certain space (film should have certain thickness and mechanical strength, prevents from subsiding) can be kept between the tissue surface be repaired and film; 5. the degradation rate of film has predictability, and degradation time and tissue regeneration process will be coordinated; 6. membrane degradation process does not hinder the healing of wound, and catabolite has no adverse reaction in organism; 7. non-immunogenicity; 8. there is certain pliability and clinical operability.Nonabsorable film is non-biodegradable materials, comprise titanium film, poly tetrafluoroethylene (e-PTFE), microporous filter membrane, cellulose membrane, extensive use is that the first two plants guiding film material clinically, wherein, titanium film is due to the attachment of the unfavorable tissue of smooth surface, enough sizes should be had during application to ensure effective barrier cell effect, allow the defective region in Mucoperiosteal flap and deep have enough contact surfaces that wound just can be avoided to split again, and there is the common complication such as wound dehiscence and film exposure after using in it; Poly tetrafluoroethylene is a kind of biomembrane that guide tissue regeneration technology adopts the earliest, its non-degradable, tissue can not be caused to be inflamed and react and immunoreation.A large amount of zoopery and clinical research confirmation e-PTFE film have pliability, good biocompatibility and the advantage such as easily to operate, desirable skeletonization effect can be ensured, but because of its nonabsorable, in clinical practice, there is more complication in e-PTFE biomembrane, as mucosa split, film exposure, infection etc.The appearance of these complication often causes skeletonization failure, and therefore nonabsorable film uses fewer and feweri clinically, has the trend replaced by Absorbable membranes.
The natural membranes material that prior art is produced is after freeze-drying radiation, natural collagen protein can significantly be destroyed, tear edge is finally caused to decline, degradation speed is accelerated, can not play good barrier action, dissimilar cell cannot be separated and grow into, and the natural membranes material that prior art is produced adds a large amount of defat, de-cell reagent, relatively large to the destruction of film, often cause the significantly reduction of the partial properties of film.In prior art, also useful cross-linking agents natural membranes material improves product tear edge and degradation resistant performance, but due to the product degradation time after crosslinked long, be not suitable for the application in Dental implantion treatment.
Summary of the invention
An object of the present invention is to provide a kind of preparation method of guide tissue regeneration film.
The preparation method of guide tissue regeneration film provided by the invention comprises the steps:
(1) use the skin of the in vitro animal of surfactant solution immersion treatment, obtain the skin after surfactant process;
(2) use the skin after surfactant process described in aqueous slkali soaking treatment step (1), obtain the dermal tissue after alkaline solution treatment;
(3) use the dermal tissue after alkaline solution treatment described in peroxide solutions immersion treatment step (2), obtain the dermal tissue after peroxide treatment;
(4) use the dermal tissue after peroxide treatment described in irradiation protection reagent solution immersion treatment step (3), obtain the dermal tissue after the process of irradiation protection reagent solution;
(5) use the dermal tissue after the process of the protection of irradiation described in buffer immersion treatment step (4) reagent solution, obtain the dermal tissue after buffer process;
(6) product after buffer process described in step (5) is carried out lyophilization and irradiation sterilization successively, obtain described guide tissue regeneration film.
In said method, in described step (4), described irradiation protection reagent is flavonoid class irradiation protective agent; Described flavonoid class irradiation protective agent can be baicalin, apigenin, kaempferol, ampelopsin, Quercetin or glycyrrhizin.
In said method, described flavonoid class irradiation protective agent is baicalin.
In said method, in described irradiation protection reagent solution, the mass fraction of described irradiation protection reagent is 0.01-0.5%.
In said method, in described step (4), the temperature of described immersion treatment is 8-20 DEG C; The time of described immersion treatment is 1-18h.
In said method,
In described step (1), described surfactant is TritonX-100, Tween-80 or Tween-40;
In described step (2), described aqueous slkali is alkali compounds aqueous solution; Described alkali compounds is NaOH or KOH; Described alkali compounds is specially NaOH;
In described step (3), described peroxide solutions is peroxide aqueous solution; Described peroxide is H
2o
2;
In described step (5), described buffer is phosphate buffer.
In said method,
The mass fraction of described surfactant is 0.1-5%;
In described alkali compounds aqueous solution, the concentration of described alkali compounds is 0.1-5mol/L;
In described peroxide aqueous solution, described H
2o
2mass fraction be 1-5%;
The pH value of described buffer is 5.0-8.0.
In said method,
In described step (1), the temperature of immersion treatment is 0-25 DEG C; The time of described immersion treatment is 20-48h;
In described step (2), the temperature of immersion treatment is 0-25 DEG C; The time of described immersion treatment is 4-18h;
The time of immersion treatment described in described step (3) is 1-5h;
The time of immersion treatment described in described step (5) is 5-48h;
In described step (6), the dosage of irradiation is 15-25KGy.
In said method, described animal is domestic animal; Described domestic animal is cattle or pig.
In said method, described domestic animal is cattle; Described cattle is the cattle of less than 12 months.
Another object of the present invention is to provide the guide tissue regeneration film that said method prepares.
Last object of the present invention is to provide the application of above-mentioned guide tissue regeneration film in the product preparing Dental implantion or auxiliary osteanagenesis or transmitting tissue's growth.
Guide tissue regeneration film prepared by the present invention have employed hypotoxicity, nonirritant abnormal flavour, residual controlled and safely and effectively natural extract flavonoid class reagent-baicalin as stablizing effective irradiation protective agent; maximize and reduce the destruction of irradiation process to the intrinsic protein structure of product, thus reduce the impact of irradiation process on product tear edge and degradation speed.Prove by experiment: guide tissue regeneration film of the present invention can be used for auxiliary osteanagenesis and transmitting tissue's growth.
Accompanying drawing explanation
Fig. 1 is the scanning electron microscope (SEM) photograph of guide tissue regeneration film prepared by the present invention.Wherein, Figure 1A is the scanning electron microscope (SEM) photograph of the guide tissue regeneration film do not obtained with the process of irradiation protective agent; Figure 1B is the scanning electron microscope (SEM) photograph of the guide tissue regeneration film obtained with the process of irradiation protective agent.
Fig. 2 is the slice map of guide tissue regeneration film.
Fig. 3 is the vivo degradation figure after subcutaneous implantation guide tissue regeneration film.
Fig. 4 is the tissue slice of bone growth situation after zoopery embedded material.
Detailed description of the invention
The experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Baicalin in following embodiment is the product of Hao Chen bio tech ltd, Shaanxi, molecular formula: C
15h
10o
5.
The preparation method of the phosphate buffer in following embodiment: claim 7.9g NaCl, 0.2g KCl, 0.24g KH
2pO
4with 1.8g K
2hPO
4, be dissolved in 800ml distilled water, regulate the pH value to 6.8 of solution with HCl, last adding distil water is settled to 1L.
The preparation of embodiment 1, guide tissue regeneration film
One, experimental group 1
1, select materials: the skin collecting fresh go out to calve (less than 12 months) by special messenger from the slaughterhouse of standardized management, avoid contact stain thing as far as possible, stored frozen immediately after collection;
2, pretreatment: after in vitro skin is thawed, it is fully cleaned, and remove the part being not suitable for processing, obtain pretreated product;
3, surfactant process: service property (quality) mark is the pretreated product of TrironX-100 aqueous solution soaking of 2%, and shaken overnight (24h), solution temperature is 0-25 DEG C, noncollagen protein composition in the pretreated product of eluting, obtains the product after surfactant process;
4, alkali treatment: the product after the NaOH aqueous solution soaking surfactant process of use 1mol/L 8 hours, solution temperature is 0-25 DEG C, makes the non-collagen albuminoid degeneration hydrolysis in the product after surfactant process, stripping, obtains the product after alkali treatment;
5, peroxide treatment: be the H of 2% with mass fraction
2o
2the product 3 hours (room temperature 25 DEG C) of aqueous solution soaking alkali treatment, obtains the product after peroxide treatment; Object is bleach product;
6, irradiation protective agent process: service property (quality) mark is the product 10 hours (8-20 DEG C) after the baicalin aqueous solution soaking peroxide treatment of 0.1%, obtains the product after the process of irradiation protective agent; Object is the destruction reducing product performance in irradiation sterilization process;
7, buffer process: soak the product 18 hours (room temperature 25 DEG C) after the process of irradiation protective agent with the phosphate buffer that pH value is 6.8, obtain the product after buffer process;
8, lyophilization: carry out lyophilizing to the product after buffer process with freezer dryer, obtains the product after lyophilization process;
9, use electron beam radiation disinfection by after the product subpackage after lyophilization process, irradiation dose is 15KGy, obtains guide tissue regeneration film after irradiation.
When other experiment conditions are identical, an experimental variable is done in this experiment in addition, and experimental variable is irradiation dose 25KGy.
Two, experimental group 2
1, select materials: the skin collecting fresh go out to calve (less than 12 months) by special messenger from the slaughterhouse of standardized management, avoid contact stain thing as far as possible, stored frozen immediately after collection;
2, pretreatment: after in vitro skin is thawed, it is fully cleaned, and remove the part being not suitable for processing, obtain pretreated product;
3, surfactant process: service property (quality) mark is the product after the Tween80 solution soaking surfactant process of 0.1%, and shaken overnight (20h), solution temperature is 0-25 DEG C, noncollagen protein composition in product after the process of eluting surface activating agent, obtains the product after surfactant process;
4, alkali treatment: the product after the NaOH solution immersion surfactant process of use 0.1mol/L 4 hours, solution temperature is 0-25 DEG C, makes the non-collagen albuminoid degeneration hydrolysis in the product after surfactant process, stripping, obtains the product after alkali treatment;
5, peroxide treatment: be the H of 1% with mass fraction
2o
2product after solution soaking alkali treatment 1 hour (room temperature 25 DEG C), obtains the product after peroxide treatment; Object is bleach product;
6, irradiation protective agent process: be the product 1 hour (8-20 DEG C) after the baicalin solution soaking peroxide treatment of 0.01% with mass fraction, obtain the product after the process of irradiation protective agent; Object is the destruction reducing product performance in irradiation sterilization process;
7, buffer process: soak the product 5 hours (room temperature 25 DEG C) after the process of irradiation protective agent with the phosphate buffer that pH value is 5, obtain the product after buffer process;
8, lyophilization: carry out lyophilizing to the product after buffer process with freezer dryer, obtains the product after lyophilization process;
9, use electron beam radiation disinfection by after the product subpackage after lyophilization process, irradiation dose is 15KGy, obtains described guide tissue regeneration film after irradiation.
When other experiment conditions are identical, an experimental variable is done in this experiment in addition, and experimental variable is irradiation dose 25KGy.
Three, experimental group 3
1, select materials: the skin collecting fresh go out to calve (less than 12 months) by special messenger from the slaughterhouse of standardized management, avoid contact stain thing as far as possible, stored frozen immediately after collection;
2, pretreatment: after in vitro skin is thawed, it is fully cleaned, and remove the part being not suitable for processing, obtain pretreated product;
3, surfactant process: service property (quality) mark is the pretreated product of Tween40 solution soaking of 5%, and shaken overnight (48h), solution temperature is 0-25 DEG C, and the noncollagen protein composition in the pretreated product of eluting, obtains the product after surfactant process;
4, alkali treatment: the product after the NaOH solution immersion surfactant process of use 5mol/L 18 hours, solution temperature is 0-25 DEG C, makes the non-collagen albuminoid degeneration hydrolysis in the product after surfactant process, stripping, obtains the product after alkali treatment;
5, peroxide treatment: be the H of 5% with mass fraction
2o
2product after solution soaking alkali treatment 5 hours (room temperature 25 DEG C), obtains the product after peroxide treatment; Object is bleach product;
6, irradiation protective agent process: be the product 18 hours (8-20 DEG C) after the baicalin solution soaking peroxide treatment of 0.5% with mass fraction, obtain the product after the process of irradiation protective agent; Object is the destruction reducing product performance in irradiation sterilization process;
7, buffer process: soak the product 48 hours (room temperature 25 DEG C) after the process of irradiation protective agent with the phosphate buffer that pH value is 8, obtain the product after buffer process;
8, lyophilization: carry out lyophilizing to the product after buffer process with freezer dryer, obtains the product after lyophilization process;
9, use electron beam radiation disinfection by after the product subpackage after lyophilization process, irradiation dose is 25KGy, obtains described guide tissue regeneration film after irradiation.
When other experiment conditions are identical, an experimental variable is done in this experiment in addition, and experimental variable is irradiation dose 15KGy.
Four, matched group 4
1, select materials: the skin collecting fresh go out to calve (less than 12 months) by special messenger from the slaughterhouse of standardized management, avoid contact stain thing as far as possible, stored frozen immediately after collection;
2, pretreatment: after in vitro skin is thawed, it is fully cleaned, and remove the part being not suitable for processing, obtain pretreated product;
3, surfactant process: be the pretreated product of TrironX-100 solution soaking of 2% with mass fraction, and shaken overnight (24h), solution temperature is 0-25 DEG C, and noncollagen protein composition in the pretreated product of eluting, obtains the product after surfactant process;
4, alkali treatment: the product after the NaOH solution immersion surfactant process of use 1mol/L 5 hours, solution temperature is 0-25 DEG C, makes non-collagen albuminoid degeneration hydrolysis, stripping in the product after surfactant process, obtains the product after alkali treatment;
5, peroxide treatment: be the H of 2% with mass fraction
2o
2product after solution soaking alkali treatment 3 hours (room temperature 25 DEG C), obtains the product after peroxide treatment; Object is bleach product;
6, buffer process: soak the product 18 hours (room temperature 25 DEG C) after peroxide treatment with the phosphate buffer that pH value is 6.8, obtain the product after buffer process;
7, lyophilization: carry out lyophilizing to the product after buffer process with freezer dryer, obtains the product after lyophilization process;
8, use electron beam radiation disinfection by after the product subpackage after lyophilization process, irradiation dose is 15KGy, obtains described guide tissue regeneration film after irradiation.
When other experiment conditions are identical, an experimental variable is done in this experiment in addition, and experimental variable is irradiation dose 25KGy.
The Performance Detection of embodiment 2, guide tissue regeneration film
1, tear edge detects
Tear edge detection method is: the guide tissue regeneration film sample that the guide tissue regeneration film obtained in the experimental group of 3 in embodiment 1 and matched group 4 prepare is cut into the strip that specification is 1cm × 3cm, sample is being passed the suture through from No. 4-0, short side edge 3-5mm place, doubling stitching thread, in distance, stitching thread is tied a knot by perforation place about 5cm place, prevents stitching thread from coming off; Then by above-mentioned sample purified water aquation 3-5 minute, one end of non-threading is fixed on the bottom of tensile testing machine, threading one end is fixed on the top of tensile testing machine by hook, start to detect, until sample is torn, reads the maximum of tensile load power, be the tear edge of this sample.Experiment in triplicate, gets the meansigma methods of 30 guide tissue regeneration films at every turn.
Result shows: compare with matched group 4, and the tear edge of guide tissue regeneration film of experimental group 1, experimental group 2 and experimental group 3 preparation all has obvious lifting, to be thisly lifted under high dosage irradiation especially obviously (table 1).
The tear edge of table 1, experimental group and matched group compares
| Irradiation metering (KGy) | Matched group 4 | Experimental group 1 | Experimental group 2 | Experimental group 3 |
| 15 | 7.38N | 9.67N | 9.35N | 9.16N |
| 25 | 5.34N | 7.85N | 7.57N | 7.12N |
2, fat content detects
Determination of fat method formulates according to the Pharmacopoeia of the People's Republic of China two " pancreatin fat detects ".
In triplicate, each 1g of guide tissue regeneration film that in Example 1, experimental group 1 and experimental group 2 obtain respectively, detect fat content, result gets the meansigma methods of three results in experiment.
Result shows: process the guide tissue regeneration film obtained and compare with coordinating through NaOH with Tween80 in experimental group 2, the guide tissue regeneration film fat content coordinating process to obtain through NaOH and TrironX-100 in experimental group 1 obviously reduces, and illustrates that the effect of TrironX-100 lipin dissolving is than Tween80 good (table 2).
Fat content after table 2, the process of different surfaces activating agent compares
| Experimental group 1 | Experimental group 2 |
| 0.08％ | 0.30％ |
3, cytotoxicity detects
4 groups of guide tissue regeneration films embodiment 1 obtained carry out Cytotoxic detection, and method detects with reference to the mtt assay in cytotoxicity detection in 16886 industry standards.In triplicate, result gets the meansigma methods of three results in experiment.
Result shows: compare with the matched group 4 in embodiment 1, and the experimental group 1 in embodiment 1, experimental group 2 and experimental group 3 decrease through the cytotoxicity of the guide tissue regeneration film that the process of irradiation protective agent obtains.Wherein, experimental group 1 reduces at most through the cytotoxicity of the guide tissue regeneration film that the process of irradiation protective agent obtains, and the cytotoxicity down ratio after process 24h and 48h is respectively 8.19% and 8.18%, and all within the scope of 0 grade (table 3).Illustrate with after the process of irradiation protective agent, the cytotoxicity of guide tissue regeneration film itself can not be improved, and cytotoxicity is had some improvement.
The cytotoxicity of table 3, experimental group and matched group compares
| Observation period (h) | Matched group 4 | Experimental group 1 | Experimental group 2 | Experimental group 3 |
| 24 | 97.31％ | 105.28％ | 100.25％ | 99.58％ |
| 48 | 99.18％ | 107.29％ | 103.49％ | 102.07％ |
4, scanning electron microscope (SEM) photograph
Fig. 1 is the scanning electron microscope (SEM) photograph of guide tissue regeneration film prepared by the present invention, and wherein Figure 1A is the scanning electron microscope (SEM) photograph of the guide tissue regeneration film that the matched group 4 in embodiment obtains without the process of irradiation protective agent; The scanning electron microscope (SEM) photograph of Figure 1B guide tissue regeneration film that to be experimental group 1 in embodiment obtain through the process of irradiation protective agent.
A large amount of irregular gap is there is between the fiber of the random braiding of collagen fiber; porosity mainly depends on the structure of natural collagen fibre; contrast known from figure; the collagen fiber of the guide tissue regeneration film obtained through the process of irradiation protective agent have certain slight shrinkage; but compare with the collagen fiber of the guide tissue regeneration film obtained without the process of irradiation protective agent and do not have a greater change, prove to use irradiation protective agent of the present invention can not destroy the original collagen fiber structure of film.
5, the slice map of guide tissue regeneration film
Fig. 2 is the slice map of the guide tissue regeneration film of experimental group 1 in embodiment 1, and as we know from the figure, guide tissue regeneration film of the present invention does not have cell and nucleus to remain, and proves that de-cell technology of the present invention effectively can slough the foreign material in membrane material.
The application of embodiment 3, guide tissue regeneration film
One, the application of guide tissue regeneration film in subcutaneous implantation
1, test method
Choose the white rabbit 12 of healthy adult, during on-test, all the weight of animals are between 1.5-3Kg, and arranging the Implantation Test phase is 1 month, 2 months, 4 months and 6 months, and operation consent animal conforms 5 days.
During implant surgery, with the sodium intravenous anesthetized animal of pentobarbital.By experimental animal accumbency operation desktop, go out back wool, routine disinfection unhairing district skin, bedding operation towel, operative region is about 80cm
2.After iodophor disinfection surgical field of view, make a kerf at back part of animal along spinal column, be about 20-50mm, blunt separation subcutaneous tissue, respectively make next subcutaneous capsule at left and right sides head end, stage casing and tail end, deeply reach superficial fascia muscle layer, each capsule spacing 20-40mm of homonymy.Each one of guide tissue regeneration film prepared by the experimental group 1 in embodiment 1 is placed in each subcutaneous capsule in left side, each one of guide tissue regeneration film sample prepared by the matched group 4 in embodiment 1 is placed in other subcutaneous capsules, sew up subcutaneous tissue and skin, povidone iodine process wound and operative region.
2, result of the test
As shown in Figure 3, wherein, Fig. 3 A is the degraded situation of guide tissue regeneration film after 1 month to vivo degradation result after subcutaneous implantation guide tissue regeneration film, and Fig. 3 B is the photo of guide tissue regeneration film degraded after 4 months.As can be seen from the figure after one month, guide tissue regeneration film is not degraded substantially, has complete film form; Guide tissue regeneration film after 4 months is degraded completely substantially, the situation and film and surrounding tissue do not stick together, surperficial smoother, and degradation time was at 3-6 month.
Two, the application of guide tissue regeneration film in Dental implantion
1, test method
Inject atropine sulfate injection 0.04mg/kg, etamsylate injection 0.5g successively to white rabbit muscle, deer sleeps peaceful 0.04-0.08mg/kg, routine disinfection, drape, pull out premolars, be separated gingiva, turn over lobe, each side certainly prepare place's box like defect according to five wall box like defect modeling methods respectively at mandibular bone, bone wound size L × W × H=15mm × 7mm × 5mm.
The guide tissue regeneration film that experimental group 1 in embodiment 1 obtains is pruned according to defect size, and is covered in defect (matched group: not coverlay material).Laboratory animal both sides gingivas is apposition suture respectively, closes wound, and intramuscular injection deer is waken up peaceful 0.04-0.08mg/kg, and postoperative intramuscular injection penicillin 800,000 units/day, compound recipe chlorhexidine collutory rinse oral cavity 5 days, carry out liquid food and raise nursing after experiment in January.
After 3 months, get alveolar bone surrounding soft tissue and osseous tissue H >=8mm, 4% formalin is fixed, and the mixing decalcifying Fluid abundant decalcification a few days, dewaters step by step, paraffin embedding, and along buccolingual diameter to doing serial section, HE dyes, om observation.
2, result of the test
Result is as shown in Figure 4: wherein, Fig. 4 A is the osteogenesis situation of matched group; Fig. 4 B is the osteogenesis situation covering guide tissue regeneration film.By finding out in slice map within the identical time, it is good that Fig. 4 B shows osteanagenesis, and in Fig. 4 A, soft tissue invades, and have impact on the normal growth of osseous tissue.Illustrate that guide tissue regeneration film of the present invention has good inducting osseous tissue regeneration performance.
Claims (10)
1. a preparation method for guide tissue regeneration film, comprises the steps:
(1) use the skin of the in vitro animal of surfactant solution immersion treatment, obtain the skin after surfactant process;
(2) use the skin after surfactant process described in aqueous slkali soaking treatment step (1), obtain the dermal tissue after alkaline solution treatment;
(3) use the dermal tissue after alkaline solution treatment described in peroxide solutions immersion treatment step (2), obtain the dermal tissue after peroxide treatment;
(4) use the dermal tissue after peroxide treatment described in irradiation protection reagent solution immersion treatment step (3), obtain the dermal tissue after the process of irradiation protection reagent solution;
(5) use the dermal tissue after the process of the protection of irradiation described in buffer immersion treatment step (4) reagent solution, obtain the dermal tissue after buffer process;
(6) dermal tissue after buffer process described in step (5) is carried out lyophilization and irradiation sterilization successively, obtain described guide tissue regeneration film.
2. method according to claim 1, is characterized in that: in described step (4), and described irradiation protection reagent is flavonoid class irradiation protective agent; Described flavonoid class irradiation protective agent is baicalin, apigenin, kaempferol, ampelopsin, Quercetin or glycyrrhizin.
3. method according to claim 1 and 2, is characterized in that: described flavonoid class irradiation protective agent is baicalin.
4., according to described method arbitrary in claim 1-3, it is characterized in that:
In described irradiation protection reagent solution, the mass fraction of described irradiation protection reagent is 0.01-0.5%;
In described step (4), the temperature of described immersion treatment is 8-20 DEG C; The time of described immersion treatment is 1-18h.
5., according to described method arbitrary in claim 1-4, it is characterized in that:
In described step (1), described surfactant is TritonX-100, Tween-80 or Tween-40;
In described step (2), described aqueous slkali is alkali compounds aqueous solution; Described alkali compounds is NaOH or KOH;
In described step (3), described peroxide solutions is peroxide aqueous solution; Described peroxide is H
2o
2;
In described step (5), described buffer is phosphate buffer.
6., according to described method arbitrary in claim 1-5, it is characterized in that:
The mass fraction of described surfactant is 0.1-5%;
In described alkali compounds aqueous solution, the concentration of described alkali compounds is 0.1-5mol/L;
In described peroxide aqueous solution, described H
2o
2mass fraction be 1-5%;
The pH value of described buffer is 5.0-8.0.
7., according to described method arbitrary in claim 1-6, it is characterized in that:
In described step (1), the temperature of immersion treatment is 0-25 DEG C; The time of described immersion treatment is 20-48h;
In described step (2), the temperature of immersion treatment is 0-25 DEG C; The time of described immersion treatment is 4-18h;
The time of immersion treatment described in described step (3) is 1-5h;
The time of immersion treatment described in described step (5) is 5-48h;
In described step (6), the dosage of irradiation is 15-25KGy.
8., according to described method arbitrary in claim 1-7, it is characterized in that: described animal is domestic animal; Described domestic animal is cattle or pig; Described domestic animal is specially cattle.
9. the guide tissue regeneration film that in claim 1-8, arbitrary described method prepares.
10. the application of guide tissue regeneration film according to claim 9 in the product preparing Dental implantion or auxiliary osteanagenesis or transmitting tissue's growth.
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