CN103893823B - A kind of high-activity biological derived material and preparation method and application - Google Patents
A kind of high-activity biological derived material and preparation method and application Download PDFInfo
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- CN103893823B CN103893823B CN201410136501.2A CN201410136501A CN103893823B CN 103893823 B CN103893823 B CN 103893823B CN 201410136501 A CN201410136501 A CN 201410136501A CN 103893823 B CN103893823 B CN 103893823B
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Abstract
The invention discloses a kind of method preparing high-activity biological derived material, comprising the steps: that (1) takes the submucosa materials of animal is raw material, is carried out and sterilizes, standby；(2) material after above-mentioned disinfecting is placed in hyperosmosis sodium chloride solution, under ultrasound condition, carry out vibration soak 1 20min, take out the material after described process and add purified water immersion, under ultrasound condition, carry out vibration soak 1 20min, thereafter letting out the water of addition, each repetitive cycling operates 46 times；(3) material after immersion treatment in above-mentioned steps (2) is carried out de-sludging and cleaning treatment, lyophilization, obtaining described high-activity biological derived material, this invention solves simple mechanical method in prior art and fails effectively to remove cellular antigens or the complicated various significantly reduced problem of biological activity causing process time length and material of processing step.
Description
Technical field
The invention belongs to medical skin tissue engineering technique field, be specifically related to a kind of high bioactivity bio-derived material
And preparation method thereof.
Background technology
Bio-derived material has the structure similar to extracellular matrix and good biocompatibility, is that one has very much
The tissue engineering bracket material of advantage.In numerous bio-derived materials, submucous layer of small intestine (SIS) has low immunogen
Property, antibiotic property and promote the advantage such as tissue growth, so being considered as a kind of ideal tissue engineering bracket material.
Containing abundant proteoglycan, collagen protein and fibronectin splicing variants in SIS, sticking between cell and tissue can be promoted
Attached, breed and break up.Proteoglycan is often at the commitment of cell adhesion, and attachment and stretching, extension stage such as cell work；Glue
Former albumen α 1 chain by type i collagen, fibronectin splicing variants is by RGD sequence and cell-membrane receptor specific bond, activation signal
Pathway promotes cell adhesion.
The multiple somatomedin that SIS comprises, such as VEGF (VEGF), basic fibroblast growth factor
Etc. (b-FGF) in the reparation and reconstruct of tissue, has important facilitation.SIS has the anatomical structure of uniqueness: collagen is fine
Heparin sulfate proteoglycan between dimension is concentrated mainly on blood vessel；In Dispersed precipitate；VEGF is then mainly distributed on around blood vessel.This
Plant structure to play a significant role in tissue regeneration.
The method processing SIS at present mainly has physical treatment process and method of chemical treatment.Physical treatment process mostly is simple machine
Division scraped by tool, i.e. strikes off the tunica muscularis intestini tenuis in addition to submucous layer of small intestine, placenta percreta and small intestinal by mechanical or manual mode viscous
Film layer three-decker, and clean by purified water or normal saline flushing.This method due to the simplest physical treatment method,
So this processing method in SIS original containing bioactive ingredients, the especially reservation of somatomedin has very well
Effect, but this method does not further remove remaining cell component in SIS, and cell component is biologically-derived material
The major antigen of material is formed, and exists so have certain immune risk when implanting.And chemical treatment method mostly is single
Pure de-cell processes and de-sludging process, some of them processing procedure is for biological active matter contained in bio-derived material
Matter impact is bigger.As disclosed in Chinese patent CN101433735A, the preparation method of a kind of SIS tissue renovation material, specifically discloses
By submucous layer of small intestine with random order disinfection, ungrease treatment, de-cell processes, de-sludging processes, after through cold
Lyophilizing is dry to be formed, and then makes soft tissue damage in skin injury, urinary system defect, mucomembranous defect and other body surface bodies
The tissue renovation material of wound, achieves process immunogenic to original material by multistep processes.But for reality in the method
Now remove antigen immune, ungrease treatment have employed substantial amounts of organic solvent, and have employed substantial amounts of surfactant
With enzyme, the bio-derived material after processing is carried out dirty removing processing.But surfactant used, organic solvent or enzyme may
The active component degeneration in bio-derived material or degraded can be caused so that bio-derived material reduces even loses biological living
Property, it is unfavorable for that bio-derived material is to skin or the reparation of tissue defect；Secondly as organic solvent and surfactant have
Certain toxicity, therefore to avoid biomaterial activity to be poisoned, needs to clean the residual organic solvent used, it is desirable to frequently
The liquid that changes process, cause the process time longer, processing shortest time is 2 days, and overall process costs increases；The more important thing is meeting
Make the biological activity of the derived material after processing by large effect.
Summary of the invention
To this end, the technical problem to be solved be in prior art the method preparing bio-derived material in order to
Remove antigen immune activity comprehensively, cause complicated process of preparation to significantly reduce asking of derived material bioactie agent content simultaneously
Topic, and then a kind of Simple process method preparing highly active bio-derived material is provided；
Second technical problem to be solved by this invention is to provide a kind of highly active bio-derived material.
For solving above-mentioned technical problem, the invention provides a kind of method preparing high-activity biological derived material, including
Following steps:
(1) tela submucosa taking animal is raw material, is carried out and sterilizes, standby；
(2) take the material after above-mentioned disinfecting to put in hyperosmosis saline solution, under ultrasound condition, carry out vibration leaching
Bubble 1-20min, takes out the material after described process and adds purified water immersion, carrying out vibration and soak 1-20min under ultrasound condition,
Thereafter let out the solution of addition, subsequently under ultrasound condition, with described hyperosmosis saline solution and water, described material is handed over
For vibration immersion treatment, each repetitive cycling operates 4-6 time；
(3) material after immersion treatment in above-mentioned steps (2) is carried out de-sludging and cleaning treatment, and will process after material
Lyophilizing, obtains described high-activity biological derived material.
The described method preparing high-activity biological derived material, in described step (2), each time described sonic oscillation soaks
The ultrasonic frequency of step is independent of each other for 10-30kHz.
The described method preparing high-activity biological derived material, in described step (2), each time described sonic oscillation soaks
The ultrasonic frequency of step is 25kHz.
The described method preparing high-activity biological derived material, in described step (2), described hyperosmosis saline solution
Mass concentration is 5-30%.
The described method preparing high-activity biological derived material, the mass concentration of described hyperosmosis saline solution is 20%.
The described method preparing high-activity biological derived material, in described step (2), described hyperosmosis saline solution
Volumetric usage is 2-5 times of described raw material weight；The volumetric usage of described purified water is the 2-5 of described raw material weight
Times.Described volumetric usage and described weight are the relation of kg/L.
The described method preparing high-activity biological derived material, in described step (2), described hyperosmosis saline solution surpasses
The time that sound soaks is 5min；The time of the ultrasonic immersion of described purified water is 5min.
The described method preparing high-activity biological derived material, in described step (1), described sterilisation step is used
Disinfectant solution be mass concentration be one of the hydrogen peroxide of 0.1-3%, 0.1-3% peracetic acid or 0.1-3% Perpropionic Acid solution and matter
Amount concentration is the mixed aqueous solution of one of ethanol or the 0.1%-15% isopropanol of 0.1%-15%.
The described method preparing high-activity biological derived material, described disinfectant solution be mass concentration be the peroxide second of 0.5%
Acid solution and the mixed aqueous solution of ethanol solution of 5%.
The described method preparing high-activity biological derived material, in described step (3), described de-sludging step is to use matter
Amount concentration is the Triton X100 of 0.5%-3%, the sodium dodecyl sulfate solution of Triton X114,0.1%-0.5% is de-sludging
Agent, carries out soaking de-sludging 1-5 time, and each de-sludging process time is 10min-20min；Described cleaning step uses normal saline to enter
Row processes.
Preferably, the described method preparing high-activity biological derived material, in described step (3), gluing of described animal
Film lower floor is submucous layer of small intestine.
The invention provides a kind of high-activity biological derived material prepared by above-mentioned method.
Present invention also offers a kind of high-activity biological derived material in the application for body-surface rauma repair materials.
The technique scheme of the present invention has the advantage that compared to existing technology
(1) method preparing high-activity biological derived material of the present invention, uses hyperosmosis under ultrasound condition
The mode that saline solution-purified water intermittent combination uses, removes the cell contained in bio-derived material, reduces its antigenicity, real
Verifying bright, the mode only with hyperosmosis-purified water Yu supersound process can effectively remove the cell that material contains, it is to avoid
Prior art needs use organic solvent and protease, nuclease etc. to remove the many deficiency that antigen causes so that this
The bio-derived material of bright preparation not only antigenicity is low, and residual that histology is acellular, DNA content is substantially less than simple mechanical
Bio-derived material prepared by method, and react without several inflammatory after implanting, overcome organic solvent and surfactant is long
Time-triggered protocol and remain excess cause bio-derived material have virose problem, have height safety and effectiveness, with
Time at utmost remain the active substance in bio-derived material so that the bioactive substance content in bio-derived material
Height, and then contribute to it to wound surface or the reparation of tissue；
(2) carry out ungrease treatment with organic solvent during the method for the invention eliminates prior art and go the step of antigen,
Prepare no matter derived material is superior to the effect of prior art in terms of biological activity or immunity, simplify preparation work
Skill；
(3) method preparing high-activity biological derived material of the present invention, optimizes the operation of ultrasonic step meticulously
Frequency parameter so that described preparation technology, on the premise of simplifying organic solvent degreasing step, still achieves ideal
Technique effect, biological property and the immune performance of gained derived material are the most even more ideal.
Accompanying drawing explanation
In order to make present disclosure be more likely to be clearly understood, below according to the specific embodiment of the present invention and combine
Accompanying drawing, the present invention is further detailed explanation, wherein
Fig. 1 is the HE colored graph of the bio-derived material of described embodiment 1 preparation；
After Fig. 2 is the bio-derived material implantation rat that prior art prepares SIS tissue renovation material and embodiment 1 preparation
The surrounding tissue inflammatory cell statistical data block diagram caused.
Detailed description of the invention
Embodiment 1
Bio-derived material described in the present embodiment is prepared in accordance with the following steps:
(1) take sheep small intestine tap water to be rinsed, then with 0.1% peracetic acid with 17% ethanol mixed aqueous solution
Process sheep small intestine, then rinse well by purified water；
(2) strike off the muscle layer of sheep small intestine, placenta percreta and the mucous layer after above-mentioned cleaning, sterilization by hand, obtain described mucosa
Lower floor 4kg, puts into described tela submucosa with in the service sink of temperature controlled ultrasonic cleaning machine automatically entering drain function, alternately uses
Hyperosmosis saline solution and purified water water carry out immersion treatment of vibrating, described ultrasonic leaching under ultrasound condition to described tela submucosa
Bubble step is as follows: entered by the hyperosmosis sodium chloride solution that the concentration of 25L is 4% in the service sink of temperature controlled ultrasonic cleaning machine, in
Tela submucosa described in ultrasonic immersion treatment under the supersonic frequency of 20kHz, discharges described hyperosmosis sodium chloride solution, so after 1min
After in described service sink, enter the purified water of 25L again, under the supersonic frequency of 30kHz under mucosa described in ultrasonic immersion treatment
Layer, discharges described purified water after 20min, described tela submucosa operates 6 according to the above-mentioned ultrasonic each repetitive cycling of immersion treatment step
Secondary；
(3) by the tela submucosa of above-mentioned steps gained with 0.3% detergent Triton X100 soaking and washing, coprocessing 6
Secondary, each 30min, then rinses described tela submucosa, by the tela submucosa of gained repeatedly with the normal saline that concentration is 0.9%
Lyophilizing, evacuation drying under reduced pressure after described material is chilled to below-40 DEG C, obtain described bio-derived material.
Embodiment 2
Bio-derived material described in the present embodiment is prepared in accordance with the following steps:
(1) take pig small intestine tap water to be rinsed, then process with the ethanol mixed aqueous solution of 3% peracetic acid with 15%
Trees-Osima jacoti, Osima excavata, then rinse well by purified water；
(2) strike off the trees-Osima jacoti, Osima excavata after above-mentioned cleaning, sterilization by hand, obtain described submucous layer of small intestine 5kg, will
Described tela submucosa is put into in the service sink of temperature controlled ultrasonic cleaning machine automatically entering drain function, alternately uses hyperosmosis chlorine
Changing sodium solution and purified water carries out immersion treatment of vibrating under ultrasound condition to described tela submucosa, described ultrasonic soaking step is such as
Under: the hyperosmosis sodium chloride solution that the mass concentration of 10L is 20% is entered in the service sink of temperature controlled ultrasonic cleaning machine, in
Tela submucosa described in ultrasonic immersion treatment under the supersonic frequency of 25kHz, discharges described hyperosmosis sodium chloride solution, so after 5min
After in described service sink, enter 10L purified water again, tela submucosa described in ultrasonic immersion treatment under the supersonic frequency of 25kHz,
Discharging described purified water after 5min, described tela submucosa operates 6 times according to the above-mentioned ultrasonic each repetitive cycling of immersion treatment step；
(3) by the tela submucosa of above-mentioned steps gained with 3% detergent Triton X114 soaking and washing, coprocessing 5 times,
20min, then repeatedly rinses described tela submucosa with the normal saline that concentration is 0.9%, the tela submucosa of gained is frozen every time
Dry, evacuation drying under reduced pressure after described material is chilled to below-40 DEG C, obtain described bio-derived material.
Embodiment 3
Bio-derived material described in the present embodiment is prepared in accordance with the following steps:
(1) take pig small intestine tap water to be rinsed, then with 3% Perpropionic Acid and 15% isopropanol mixed aqueous solution at
Reason pig small intestine, then rinse well by purified water；
(2) strike off the trees-Osima jacoti, Osima excavata after above-mentioned cleaning, sterilization by hand, obtain described submucous layer of small intestine 0.5kg,
Described submucous layer of small intestine is put into in the service sink of temperature controlled ultrasonic cleaning machine automatically entering drain function, alternately ooze with height
Pressure sodium chloride solution and purified water carry out immersion treatment of vibrating, described ultrasonic immersion under ultrasound condition to described tela submucosa thoroughly
Step is as follows: entered by the hyperosmosis sodium chloride solution that the concentration of 1.5L is 30% in the service sink of temperature controlled ultrasonic cleaning machine, in
Submucous layer of small intestine described in ultrasonic immersion treatment under the supersonic frequency of 30kHz, discharges described hyperosmosis sodium chloride molten after 20min
Liquid, enters 1.5L purified water the most again in described service sink, small intestinal described in ultrasonic immersion treatment under the supersonic frequency of 10kHz
Tela submucosa, discharges described purified water after 1min, described submucous layer of small intestine respectively repeats according to above-mentioned ultrasonic immersion treatment step
Circulation operation 5 times；
(3) submucous layer of small intestine of above-mentioned steps gained is soaked clear with the detergent sodium dodecyl sulfate solution of 0.5%
Wash, coprocessing 2 times, each 15min, then repeatedly rinse described tela submucosa, by gained with the normal saline that concentration is 0.9%
Tela submucosa lyophilizing, evacuation drying under reduced pressure after described material is chilled to below-40 DEG C, obtain described biologically-derived material
Material.
Embodiment 4
Bio-derived material described in the present embodiment is prepared in accordance with the following steps:
(1) take pig small intestine tap water to be rinsed, then with 0.1% hydrogen peroxide and the isopropanol mixing water of 0.1%
Solution processes pig small intestine, then rinses well by purified water；
(2) strike off the muscle layer of pig small intestine, placenta percreta and the mucous layer after above-mentioned cleaning, sterilization by hand, obtain described mucosa
Lower floor 1kg, puts into described tela submucosa with in the service sink of temperature controlled ultrasonic cleaning machine automatically entering drain function, alternately uses
Hyperosmosis sodium chloride solution and purified water carry out immersion treatment of vibrating under ultrasound condition to described tela submucosa, described ultrasonic
Soaking step is as follows: the hyperosmosis sodium chloride solution that the mass concentration of 5L is 5% enters the service sink of temperature controlled ultrasonic cleaning machine
In, tela submucosa described in ultrasonic immersion treatment under the supersonic frequency of 10kHz, discharge described hyperosmosis sodium chloride after 10min
Solution, enters 5L purified water the most again in described service sink, mucosa described in ultrasonic immersion treatment under the supersonic frequency of 30kHz
Lower floor, discharges described purified water after 10min, described tela submucosa operates according to the above-mentioned ultrasonic each repetitive cycling of immersion treatment step
4 times；
(3) by the detergent Triton X100 soaking and washing that tela submucosa concentration is 0.5% of above-mentioned steps gained, instead
Process 1 time again, each 10min, then repeatedly rinse described tela submucosa with the normal saline that concentration is 0.9%, gluing gained
Film subsurface material carries out frozen dried, evacuation drying under reduced pressure after described material is chilled to below-40 DEG C, obtains described biology
Derived material.
Embodiment 5
Bio-derived material described in the present embodiment is prepared in accordance with the following steps:
(1) take sheep small intestine tap water to be rinsed, then with 3% Perpropionic Acid and the ethanol mixed aqueous solution of 0.1%
Process sheep small intestine, then rinse well by purified water；
(2) strike off the muscle layer of sheep small intestine, placenta percreta and the mucous layer after above-mentioned cleaning, sterilization by hand, obtain described mucosa
Lower floor 1.5kg, puts into described tela submucosa with in the service sink of temperature controlled ultrasonic cleaning machine automatically entering drain function, replaces
By hyperosmosis sodium chloride solution and purified water, described tela submucosa is carried out immersion treatment of vibrating under ultrasound condition, described super
Sound soaking step is as follows: the hyperosmosis sodium chloride solution that the concentration of 3L is 25% enters the service sink of temperature controlled ultrasonic cleaning machine
In, tela submucosa described in ultrasonic immersion treatment under the supersonic frequency of 28kHz, discharge described hyperosmosis sodium chloride after 15min
Solution, enters 3L purified water the most again in described service sink, mucosa described in ultrasonic immersion treatment under the supersonic frequency of 15kHz
Lower floor, discharges described purified water after 1min, described tela submucosa operates 5 according to the above-mentioned ultrasonic each repetitive cycling of immersion treatment step
Secondary；
(3) by the tela submucosa of above-mentioned steps gained with 0.1% detergent sodium dodecyl sulfate solution soaking and washing,
Coprocessing 3 times, each 13min, then rinses described tela submucosa with the normal saline that concentration is 0.9%, gluing gained repeatedly
The lyophilizing of film lower floor, evacuation drying under reduced pressure after described material is chilled to below-40 DEG C, obtain described bio-derived material.
Effect example
Prepared by present invention bio-derived material and in the prior art SIS of preparation is better described below by way of effect example
Tissue renovation material has essentially identical safety and effectiveness, and bio-derived material prepared by the present invention is the most at utmost
Remain bioactive substance, and then obtain the notable skill that described bio-derived material is high to the biological activity of wound surface or tissue
Art effect.
1. experiment material
Experiment material: the bio-derived material of the embodiment of the present invention 1 preparation and employing prior art Patent Literature
The SIS tissue renovation material that in CN101433735A, embodiment 1 prepares.
2. experimental technique and result
2.1 safeties and the detection of effectiveness
Respectively by the SIS group of preparation in the bio-derived material of the embodiment of the present invention 1 and patent documentation CN101433735A
Knitting repair materials and carry out HE dyeing process, it is as follows that described HE dyeing processes step: by descending for material graded ethanol enter water, use afterwards
Tap water and distilled water successively clean, and clean with tap water, then use with after 1% hydrochloride alcohol color separation after processing with 0.5% haematoxylin
Tap water cleans, then promotees to clean after basket with the ammonia of 1/400, with after 1% eosin stains with 80% ethanol color separation, graded ethanol is de-
Water, obtains resin mounting after dimethylbenzene is transparent.The HE colored graph of the bio-derived material of the embodiment of the present invention 1 preparation is shown in Fig. 1, says
The comparison of the bio-derived material cell removing of the bright embodiment of the present invention 1 preparation is clean, has no the intact cell configuration of residual.
The SIS of preparation in the detection bio-derived material of the embodiment of the present invention 1 and patent documentation CN101433735A respectively
DNA remaining quantity in tissue renovation material, described DNA content detection method is as follows: (1), by material 37 DEG C, disappears with E.C. 3.4.21.64
Change 144 hours；(2) by Digestive system with centrifugal 30 minutes；(3) by supernatant phenol chloroform-isoamyl alcohol (25:24:1)
Extracting and purifying；(4) extract is centrifuged 30 minutes again, removes upper water solution；(5) add ethanol, be placed in-20 DEG C and preserve 8
More than hour；(6) vacuum dehydration DNA sample；(7) by 1xTE buffer solution DNA sample；(8) preparation 2 × PicoGreen
Reagent working solution: with PH=8.01 × TE, concentrate PicaGreen by the dilution proportion of 1:200；(9) configuration standard product solution testing
And obtain concentration curve, and demarcate DNA content curve, excitation spectrum wavelength is 485nm bandwidth 20nm, and emission spectrum wavelength is
530nm bandwidth 25nm；(10) test sample fluorescent value, calculates its DNA concentration according to sample fluorescence value, and converted dna accounts for material
The percentage composition of gross weight.Detection obtains, and the DNA content of the bio-derived material of the embodiment of the present invention 1 is 18.1 ± 8.9ng/mg,
And the DNA content in the SIS tissue renovation material of preparation is 44.7 ± 10.4ng/mg in patent documentation CN101433735A, two
Person compares discovery, and the ratio of the donorcells removing of bio-derived material prepared by the present invention more thoroughly, illustrates side of the present invention
Method can effectively remove cell, and the antigenicity of the bio-derived material of preparation is relatively low, and safety is high.
Respectively by the SIS group of preparation in the bio-derived material of the embodiment of the present invention 1 and patent documentation CN101433735A
Knitting repair materials to implant in healthy SD rat, described Implantation Test method is as follows: take 20 male SD rats, 3-4 week old, body weight
180-220g, is subcutaneously implanted in patent documentation CN101433735A the material of preparation after anesthesia, implant the present invention at offside and prepare
Material, every material 1cm2, randomly select implantation 1 week respectively, 2 weeks, 4 weeks, after 8 weeks, each 5 rats were put to death, described rat
Lesion is used for inflammatory reaction evaluation, and the surrounding tissue paraformaldehyde taking described lesion is fixed, specimens paraffin embedding slices, and HE dyes,
Take 6 visual field photograph at random in every tangent plane, carry out inflammatory cell, including mononuclear cell, polymorphonuclear leukocyte number, pass through
Digital image analysis system counting processes, unit of account area (mm2) inflammatory cell density, compare after two groups of materials are implanted and cause
The situation of surrounding tissue inflammatory reaction, described statistics block diagram is shown in Fig. 2, it was found that two groups of material inflammatory reaction in implanting 4 weeks
Broadly fall into comparatively gentle inflammatory reaction, but inflammatory cell density is slightly different and does not has significant difference, two groups of materials after 8 weeks
Around implanting tissue, inflammatory reaction the most almost disappears, and finds that described bio-derived material almost all is absorbed by tissue, and
The autologous cambium that its induction grows without obvious boundary and plesiomorphism with autologous tissue's structure, is not easily distinguishable, has no lymph
Cellular infiltration, does not finds serious inflammatory reaction, illustrates that material prepared by the bio-derived material of the present invention and prior art exists
Difference is not had in safety.
The mensuration of 2.2 bioactive substances
Bio-derived material and the patent documentation of the embodiment of the present invention 1 is detected respectively by ELISA method
The growth factor content of the SIS tissue renovation material of preparation in CN101433735A.ELISA method of testing key step: in training
Support in plate the VEGF(ng/mL adding normal concentration) and standard substance b-FGF(pg/mL), patent documentation CN101433735A in
The SIS tissue renovation material of preparation and the bio-derived material of the embodiment of the present invention 1, then to patent documentation CN101433735A
The SIS tissue renovation material of middle preparation and the bio-derived material of the embodiment of the present invention 1 add biotin, adds in each sample
Enter enzyme labelling liquid, mix latter 37 DEG C and hatch 1h, wash plate 5 times, each 5min, then in each test specimens, add substrate in right amount, mixing
Hatch 15min, be eventually adding stop buffer, put into test instrunment and detect for latter 37 DEG C.Measurement result is, the embodiment of the present invention 1
Bio-derived material in growth factor VEGF content be 52.2 ± 7.5ng/mg, bFGF content be 4.6 ± 0.6pg/mg, and
The growth factor VEGF content 70.7 ± 15.9ng/mg in submucous layer of small intestine described after striking off by hand, bFGF content 5.4 ±
1.7pg/mg.And the growth factor VEGF content in the SIS tissue renovation material of preparation is in patent documentation CN101433735A
27.4 ± 6.1ng/mg, bFGF content is 3.2 ± 1.6pg/mg, the growth in submucous layer of small intestine described after striking off by hand because of
Sub-VEGF content 75.4 ± 25.3ng/mg, bFGF content 6.0 ± 4.7pg/mg, illustrate to make in patent documentation CN101433735A
Standby the growth factors such as VEGF content in SIS tissue renovation material, bFGF content are far smaller than and strike off rear gained with manual
Above-mentioned growth factor content in submucous layer of small intestine, although and heretofore described bio-derived material strikes off rear institute than manual
Growth factor content in the submucous layer of small intestine obtained decreases, but compares preparation in patent documentation CN101433735A
Growth factor content in SIS tissue renovation material is significantly raised, preferably remains the biological activity of submucous layer of small intestine.
3. conclusion
(1) bio-derived material prepared by the present invention and the safety of the SIS tissue renovation material of preparation in prior art
Suitable with effectiveness；
(2) bioactive substance content in bio-derived material prepared by the present invention is far above preparation in prior art
Bioactive substance content in SIS tissue renovation material.
Obviously, above-described embodiment is only for clearly demonstrating example, and not restriction to embodiment.Right
For those of ordinary skill in the field, can also make on the basis of the above description other multi-form change or
Variation.Here without also cannot all of embodiment be given exhaustive.And the obvious change thus extended out or
Change among still in the protection domain of the invention.
Claims (11)
1. the method preparing high-activity biological derived material, it is characterised in that comprise the steps:
(1) tela submucosa taking animal is raw material, is carried out and sterilizes, standby, and what wherein said sterilisation step was used disappears
Venom be mass concentration be one of hydrogen peroxide or 0.1-3% Perpropionic Acid solution of 0.1-3% be 0.1%-15% with mass concentration
The mixed aqueous solution of isopropanol；
(2) take the material after above-mentioned disinfecting to put in hyperosmosis saline solution, under ultrasound condition, carry out vibration soak 1-
20min, takes out the material after described process and adds purified water immersion, carrying out vibration and soak 1-20min under ultrasound condition, with
The rear solution discharging addition, is carried out at alternately sonic oscillation immersion described material with described hyperosmosis saline solution and purified water
Reason, each repetitive cycling operates 4-6 time；The volumetric usage of described hyperosmosis saline solution is 2-5 times of described raw material weight；
The volumetric usage of described purified water is 2-5 times of described raw material weight；Described volumetric usage and described weight are kg/L's
Relation；The ultrasonic frequency of above-mentioned each described sonic oscillation soaking step is 10-30kHz；Described hyperosmosis saline solution
Mass concentration is 5-30%；
(3) material after immersion treatment in above-mentioned steps (2) being carried out de-sludging and cleaning treatment, described de-sludging step is to use matter
Amount concentration is the Triton X100 of 0.5%-3%, Triton X114 or 0.1-0.5% sodium dodecyl sulfate solution is detergent,
Carrying out soaking de-sludging 1-5 time, each de-sludging process time is 10min-20min；Described cleaning step uses normal saline to carry out
Process, and will process after material lyophilizing, obtain described high-activity biological derived material.
The method preparing high-activity biological derived material the most according to claim 1, it is characterised in that described step (2)
In, the ultrasonic frequency of each described sonic oscillation soaking step is 25kHz.
The method preparing high-activity biological derived material the most according to claim 1 and 2, it is characterised in that described height oozes
The mass concentration of pressure saline solution is 20% thoroughly.
The method preparing high-activity biological derived material the most according to claim 3, it is characterised in that described step (2)
In, the time of the described ultrasonic immersion of hyperosmosis saline solution is 5min；The time of the ultrasonic immersion of described purified water is 5min.
5. according to the method preparing high-activity biological derived material described in claim 1 or 2 or 4, it is characterised in that described in disappear
Venom be mass concentration be peracetic acid soln and the mixed aqueous solution of ethanol solution of 5% of 0.5%.
The method preparing high-activity biological derived material the most according to claim 5, it is characterised in that described step (1)
In, the tela submucosa of described animal is submucous layer of small intestine.
The method preparing high-activity biological derived material the most according to claim 1, it is characterised in that include walking as follows
Rapid:
(1) take pig small intestine tap water to be rinsed, then process pig with the isopropanol mixed aqueous solution of 3% Perpropionic Acid and 15%
Small intestinal, then rinse well by purified water；
(2) strike off the trees-Osima jacoti, Osima excavata after above-mentioned cleaning, sterilization by hand, obtain described submucous layer of small intestine 0.5kg, by institute
State submucous layer of small intestine to put into in the service sink of temperature controlled ultrasonic cleaning machine automatically entering drain function, alternately use hyperosmosis
Sodium chloride solution and purified water carry out immersion treatment of vibrating, described ultrasonic soaking step under ultrasound condition to described tela submucosa
As follows: the hyperosmosis sodium chloride solution that the mass concentration of 1.5L is 30% to be entered in the service sink of temperature controlled ultrasonic cleaning machine, in
Submucous layer of small intestine described in ultrasonic immersion treatment under the supersonic frequency of 30kHz, discharges described hyperosmosis sodium chloride molten after 20min
Liquid, enters 1.5L purified water the most again in described service sink, small intestinal described in ultrasonic immersion treatment under the supersonic frequency of 10kHz
Tela submucosa, discharges described purified water after 1min, described submucous layer of small intestine respectively repeats according to above-mentioned ultrasonic immersion treatment step
Circulation operation 5 times；
(3) by the submucous layer of small intestine of above-mentioned steps gained with 0.5% detergent sodium dodecyl sulfate solution soaking and washing,
Coprocessing 2 times, each 15min, then rinses described tela submucosa with the normal saline that concentration is 0.9%, gluing gained repeatedly
The lyophilizing of film lower floor, evacuation drying under reduced pressure after described material is chilled to below-40 degree, obtain described bio-derived material.
The method preparing high-activity biological derived material the most according to claim 1, it is characterised in that include walking as follows
Rapid:
(1) take pig small intestine tap water to be rinsed, then with 0.1% hydrogen peroxide and the isopropanol mixed aqueous solution of 0.1%
Process pig small intestine, then rinse well by purified water；
(2) strike off the muscle layer of pig small intestine, placenta percreta and the mucous layer after above-mentioned cleaning, sterilization by hand, obtain described tela submucosa
1kg, puts into described tela submucosa with in the service sink of temperature controlled ultrasonic cleaning machine automatically entering drain function, alternately oozes with height
Pressure sodium chloride solution and purified water carry out immersion treatment of vibrating, described ultrasonic immersion under ultrasound condition to described tela submucosa thoroughly
Step is as follows: entered by the hyperosmosis sodium chloride solution that the mass concentration of 5L is 5% in the service sink of temperature controlled ultrasonic cleaning machine,
Tela submucosa described in ultrasonic immersion treatment under the supersonic frequency of 10kHz, discharges described hyperosmosis sodium chloride molten after 10min
Liquid, enters 5L purified water, under the supersonic frequency of 30kHz under mucosa described in ultrasonic immersion treatment the most again in described service sink
Layer, discharges described purified water after 10min, described tela submucosa operates 4 according to the above-mentioned ultrasonic each repetitive cycling of immersion treatment step
Secondary；
(3) by the detergent Triton X100 soaking and washing that tela submucosa concentration is 0.5% of above-mentioned steps gained, repeatedly locate
Manage 1 time, each 10min, then repeatedly rinse described tela submucosa, by under the mucosa of gained with the normal saline that concentration is 0.9%
Layer material carries out frozen dried, evacuation drying under reduced pressure after described material is chilled to below-40 degree, obtains described biologically-derived
Material.
The method preparing high-activity biological derived material the most according to claim 1, it is characterised in that include walking as follows
Rapid:
(1) take sheep small intestine tap water to be rinsed, then process with the Perpropionic Acid of 3% and the ethanol mixed aqueous solution of 0.1%
Sheep small intestine, then rinse well by purified water；
(2) strike off the muscle layer of sheep small intestine, placenta percreta and the mucous layer after above-mentioned cleaning, sterilization by hand, obtain described tela submucosa
1.5kg, puts into in the service sink of temperature controlled ultrasonic cleaning machine automatically entering drain function, alternately with height by described tela submucosa
Isotonic sodium chloride solution and purified water carry out immersion treatment of vibrating, described ultrasonic leaching under ultrasound condition to described tela submucosa
Bubble step is as follows: the hyperosmosis sodium chloride solution that the mass concentration of 3L is 25% enters the service sink of temperature controlled ultrasonic cleaning machine
In, tela submucosa described in ultrasonic immersion treatment under the supersonic frequency of 28kHz, discharge described hyperosmosis sodium chloride after 15min
Solution, enters 3L purified water the most again in described service sink, mucosa described in ultrasonic immersion treatment under the supersonic frequency of 15kHz
Lower floor, discharges described purified water after 1min, described tela submucosa operates 5 according to the above-mentioned ultrasonic each repetitive cycling of immersion treatment step
Secondary；
(3) by the tela submucosa of above-mentioned steps gained by the detergent sodium dodecyl sulfate solution soaking and washing of 0.1%, coexist
Manage 3 times, each 13min, then repeatedly rinse described tela submucosa, by under the mucosa of gained with the normal saline that concentration is 0.9%
Layer lyophilizing, evacuation drying under reduced pressure after described material is chilled to below-40 degree, obtain described bio-derived material.
10. the high-activity biological derived material prepared according to the arbitrary described method of claim 1-9.
High-activity biological derived material described in 11. claim 10 is for the application of body-surface rauma repair materials.
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| CN105664256A (en) * | 2016-02-23 | 2016-06-15 | 江苏期佰医疗技术有限公司 | Method for preparing restoring material with sustained release medicine |
| CN116942913B (en) * | 2023-07-26 | 2024-02-27 | 江苏博朗森思医疗器械有限公司 | Acellular matrix material and preparation method and application thereof |
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| CA2736663A1 (en) * | 2007-09-07 | 2009-03-12 | Surgical Biologics, Llc. | Placental tissue grafts and improved methods of preparing and using the same |
| CN101433735A (en) * | 2007-11-13 | 2009-05-20 | 北京大清生物技术有限公司 | Method for preparing SIS tissue repair material |
| CN102218162A (en) * | 2011-05-24 | 2011-10-19 | 山西奥瑞生物材料有限公司 | Preparation method of homologous acellular dermal matrix |
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| CN103251987A (en) * | 2013-04-07 | 2013-08-21 | 陕西佰傲再生医学有限公司 | Acellular biological patch, preparation method and apparatus thereof |
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| US20060134072A1 (en) * | 2004-12-22 | 2006-06-22 | Pedrozo Hugo A | Self-assembling protein matrix prepared from natural extracellular matrices |
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|---|---|---|---|---|
| CA2736663A1 (en) * | 2007-09-07 | 2009-03-12 | Surgical Biologics, Llc. | Placental tissue grafts and improved methods of preparing and using the same |
| CN101433735A (en) * | 2007-11-13 | 2009-05-20 | 北京大清生物技术有限公司 | Method for preparing SIS tissue repair material |
| CN102225218A (en) * | 2011-04-26 | 2011-10-26 | 中国人民解放军第三军医大学第一附属医院 | Method for preparing acellular dermal matrix by utilizing ultrasonic wave |
| CN102218162A (en) * | 2011-05-24 | 2011-10-19 | 山西奥瑞生物材料有限公司 | Preparation method of homologous acellular dermal matrix |
| CN103251987A (en) * | 2013-04-07 | 2013-08-21 | 陕西佰傲再生医学有限公司 | Acellular biological patch, preparation method and apparatus thereof |
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