CN108982769A - A kind of biocompatibility detection method of collagen protein sponge - Google Patents
A kind of biocompatibility detection method of collagen protein sponge Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
Abstract
The invention discloses a kind of biocompatibility detection methods of collagen protein sponge, comprising the following steps: (1) preparation of collagen protein sponge leaching liquor；(2) Sterility testing of collagen protein sponge；(3) detection of bacterial endotoxin of collagen protein sponge；(4) the systemic acute toxi-city detection of collagen protein sponge；(5) the cytotoxicity detection of collagen protein sponge；(6) the hemolytic test of collagen protein sponge；(7) the hypersensitive test of collagen protein sponge；(8) picosecond laser pulse of collagen protein sponge；(9) Implantation Test of collagen protein sponge；(10) genetic toxicity test of collagen protein sponge；(11) the degradation detection of collagen protein sponge.Comprehensively, effectively, gained qualified products meet national legislation standard to this method, have the effect of excellent promotion wound healing and good product quality.
Description
Technical field
The present invention relates to a kind of biocompatibility detection methods of sponge, belong to pharmaceutical technology field, in particular to a kind of
The biocompatibility detection method of collagen protein sponge.
Background technique
Due to the development of biochemistry, molecular biology and cytobiology technology, people to extracellular matrix, particularly
To its main component --- the interest of collagen is increasingly dense, the understanding of research method and structure is gradually increased, especially
Clinically using more and more extensive, especially stop blooding in surgery wound and repair, radical cure fistula, remove ulcerated tissue and infectivity
Aspect all has received good efficacy.
In the world many developed countries at present all in research, exploitation, utilize collagen.Especially to its medical value
Research is developed, using attaching the importance, wherein being maintained the leading position with the U.S..Many scientific research departments of China are also energetically by glue
Former albumen is in daily life.Such as medical collagen is developed for treating disease and beauty at medical aspect.Cause
This collagen comes into our daily life.
In many countries uses quite extensively, especially in terms of clinical medicine, product is to various for collagen product
The treatment of wound provides great convenience, the work for having fully demonstrated its hemostasis, having promoted hyperblastosis, accelerating wound healing
With especially to the chronic wound for being difficult to heal, such as the treatment of fistula, sinus, bedsore, it is shown that its superiority.
Various wound hemostasis are in medical industry and the hot spot of medical field research.It is also in various wounds and surgical procedure
The therapeutic process of doctor's emphasis, using there is biology to mix, the hemostatic material of absorbable and degradable is safety in the hemostasis of the various surface of a wound
Effectively, easy to use that there are bright prospects, it benefits the nation and the people, the major issue of healthy human.But there is not collagen protein sponge tool yet at present
The biocompatibility detection method of body.
Summary of the invention
For overcome the deficiencies in the prior art, it is an object of the present invention to provide a kind of inspections of the biocompatibility of collagen protein sponge
Survey method, comprehensively, effectively, gained qualified products meet national legislation standard to this method, with excellent promotion wound healing
Effect and good product quality.
In order to achieve the above object, the invention adopts the following technical scheme:
The present invention provides a kind of biocompatibility detection method of collagen protein sponge, comprising the following steps:
(1) preparation of collagen protein sponge leaching liquor；
(2) Sterility testing of collagen protein sponge；
(3) detection of bacterial endotoxin of collagen protein sponge；
(4) the systemic acute toxi-city detection of collagen protein sponge；
(5) the cytotoxicity detection of collagen protein sponge；
(6) the hemolytic test of collagen protein sponge；
(7) the hypersensitive test of collagen protein sponge；
(8) picosecond laser pulse of collagen protein sponge；
(9) Implantation Test of collagen protein sponge；
(10) genetic toxicity test of collagen protein sponge；
(11) the degradation detection of collagen protein sponge.
Further, in step (1), the preparation of the leaching liquor are as follows: by collagen protein sponge matching by 0.5-2cm/mL
Than being impregnated 24-48 hours with physiological saline.
Further, in step (2), the Sterility testing are as follows: the THIOGLYCOLLIC ACID salt broth of test sample will be inoculated with
Container be divided into two equal parts, portion sets 30~35 DEG C of cultures, and portion sets 20~25 DEG C of cultures, should observe and remember day by day during culture
Whether record has bacterium growth.
Further, in step (3), the detection of bacterial endotoxin are as follows: 4, the test tube equipped with reagents solution, wherein 2
0.1mL test sample is added as test product pipe in branch, and 12 λ endotoxin working standard 0.1ml of addition is as positive control pipe, and 1
The reagents solution 0.1mL worn is added as negative control pipe, after test tube is mixed gently, closes nozzle, is vertically put into 37
In ± 1 DEG C of water-bath, 1-2h is kept the temperature.
Further, in step (4), the systemic acute toxi-city detection are as follows: selecting healthy mice even numbers, only weight exists
17-23g, male and female are each flat, and mean random is divided into experimental group and control group, and leaching liquor is taken to carry out abdominal cavity note to experimental group small white mouse
It penetrates, control group injecting normal saline, the upper and lower noon is each primary, and injection dosage is 0.2-0.6mL/10g weight, continuous 7 days, observes
Its sign situation.
Further, in step (5), the cytotoxicity detection are as follows: using MTT colorimetric determination collagen protein sponge
Influence of the leaching liquor to 2,4,7 days opposite proliferation rates of fibroblast, evaluates the cytotoxicity of collagen protein sponge.
Preferably, collagen protein sponge 1cm to be measured210mL cell culture fluid is added, sets 37 DEG C of standings and takes supernatant in 24 hours
Material leaching liquor of the liquid as 100% concentration；Cell culture fluid doubling dilution is used again, and it is spare that 50% material leaching liquor is made.
Preferably, the measurement of cell opposite proliferation rate and Materials Cell toxicity assessment: with cell culture fluid logarithmic growth phase
It is about 5-8 × 10 that Mouse Skin Fibroblasts (2-3 generation), which are made into concentration,4A/mL cell suspension, every 100 μ L of hole, is inoculated in
Culture plate sets 37 DEG C, 5%CO2It is cultivated 24-48 hours in incubator；It is replaced respectively with the material leaching liquor of 50%, 100% concentration
(200 hole μ L/) is changed, using cell culture fluid as blank control, continues to overturn away liquid in 2 holes after setting 37 DEG C of cultures 3-6 hours,
Every hole adds DMSO 150-200 μ L, shakes 10-20min；Enzyme linked immunological instrument measures the absorbance value in every hole, and each group 8 take mean value；
Measurement wavelength is 490nm；The cell opposite proliferation rate of sample is calculated by formula R=experimental group OD/ control group OD × 100% later
(RGR) the cytotoxicity classification of collagen protein sponge is evaluated.
Further, in step (6), the hemolytic test are as follows: take rabbit heart blood 10-20mL, separating red corpuscle is made
Volume fraction is that 2%RBC suspension is spare；Test is divided into test product pipe, negative control pipe and positive control pipe, and every group sets 4-8
Parallel pipe；Developmental tube is dilution of each concentration leaching liquor to physiological saline, and negative control uses physiological saline, and positive control is adopted
Use distilled water；Each pipe is added 8-20mL respective sample, and after 37 DEG C of water-soluble 30-60min, respectively plus the RBC of 0.16-0.4mL is suspended
Liquid is stood overnight after 37 DEG C of water-soluble 1-2h, centrifuged supernatant colorimetric at 520cm.
Further, in step (7), hypersensitive test are as follows: take healthy guinea pig even numbers only, the next day be injected intraperitoneally
0.15-0.5mL leaching liquor, continuous 3 times, wherein half cavy in for the first time inject after continuous 14 days by be injected intraperitoneally 1.5-2.5mL
Leaching liquor；In addition half cavy 21 days intraperitoneal injection 1.5-2.5mL leaching liquors after injecting for the first time；15 points are observed after per injection
Have after clock, especially final injection it is useless grab scratch nose, sneeze, perpendicular hair, twitch, expiratory dyspnea, gatism, body temperature change,
The reaction such as shock, death.
Further, in step (8), the picosecond laser pulse are as follows: using rabbit as object, right hindlimb quadriceps muscle of thigh is
Test area injects 1-2mL leaching liquor, and left side corresponding position is injected isometric physiological saline with method and compared, and injection 48 is small
When, 14 days, grouping in 21 days put to death test rabbit, dissection take out quadriceps muscle of thigh, it is longitudinally slit, observation injection site musculature become
Change.
Further, in step (9), the Implantation Test are as follows: take male mouse of kunming, weight 25-30g；Aseptic condition
Under, collagen protein sponge is made to the former piece of diameter 1cm of punch；It is fixed after mouse anesthesia, the disinfection of back center, unhairing,
Collagen protein sponge is put into right side subcutaneously by the stringer notch for cutting off about 0.7cm or so, normal to raise after 2 needle of catgut suture；
Postoperative 3,5,7,9 natural gift other places, which after death visually observe and draw materials, carries out routine histologic inspection.
Further, in step (10), the genetic toxicity test are as follows: gene mutation is detected by Salmonella reversion test；Pass through
Micronucleus test detects chromosome aberration；It is distorted by chromosome aberrations in spermatgognium testing inspection genome；By unicellular
Gel electrophoresis analysis detects DNA initial injury.
Further, in step (11), the degradation detection are as follows: starch is placed in and contains at a temperature of 37 DEG C ± 2 DEG C in product
Have in the physiological saline of a- amylase and carbohydrase after 72-96 hours, under a- amylase and saccharification enzyme effect, is converted into grape
Sugar, conversion ratio are equal to degradation rate.
Further, the biocompatibility detection should meet following index:
The collagen protein sponge is sterile after Co-60 ray sterilizing；
The bacterial endotoxin reaches≤0.5EU/mg；
The collagen protein sponge will not cause acute poisoning symptom；
The cytotoxicity reaches≤1 grade；
The collagen protein sponge does not cause hemolytic reaction；
The collagen protein sponge does not lead to body allergic reaction；
The collagen protein sponge has no stimulation to deep tissue, has no apparent inflammatory reaction, does not generate apparent
Tissue reaction；
The collagen protein sponge is after muscular grafting 7 days, the inflammatory cell extent of reaction≤IV grades；It is scorching after implantation 15 days
Disease cell effect degree≤III level；Implantation 30 days after, the inflammatory cell extent of reaction≤II grade, and around be no different paradoxical reaction 6 weeks after
It is absorbed；
The collagen protein sponge hereditary-less toxicity.
Compared with prior art, the invention has the following advantages:
The biocompatibility detection method of collagen protein sponge of the invention comprehensively, effectively, clearly provides the inspection of each index
Survey method and result standard, gained qualified products meet national legislation standard, have the effect of excellent promotion wound healing and
Good product quality.
Specific embodiment
Now in conjunction with specific embodiment, the invention will be further described.
The present invention is applicable in germ-free condition supply and disposable absorbability collagen haemostatic sponge, the product
In to capillary, vein and artery hemostasis and diffusivity bleeding, various wounds and all kinds of surgical wound surfaces can be widely used in
Hemostasis.
Absorbability collagen haemostatic sponge is that one kind is applied to the bleeding surface of a wound, the medical material with hemostasia effect.
Biocompatibility is the extremely important performance of biomedical material, to ensure safety of the materials'use after human body
Property, the extracting solution of the collagen protein sponge of our company's production has been subjected to acute toxicity test, irritation test, anaphylaxis examination
It tests with hemolytic test, cell toxicity test and has carried out subcutaneous implant test with collagen protein sponge.
The preparation of 1 leaching liquor of embodiment
The proportion that collagen protein sponge is pressed to 1.25cm/mL is impregnated 24 hours with physiological saline, obtains extracting solution.
2 Sterility testing of embodiment
The container for being inoculated with the THIOGLYCOLLIC ACID salt broth of test sample is divided into two equal parts, and portion sets 30~35 DEG C of cultures,
Portion sets 20~25 DEG C of cultures.Whether have bacterium growth, such as test sample is being added or is training if should be observed and recorded day by day during culture
During supporting, there is muddiness in culture medium, after culture 14 days, cannot determine whether microorganism growth from the appearance, can use the culture
The appropriate transferred species of liquid is cultivated 3 days, whether the fresh culture of the same race for observing inoculation muddiness occurs again into fresh culture of the same race；
Or culture solution smear is taken, it dyes, microscopy judges whether there is bacterium.
As a result: product is sterile after Co-60 ray sterilizing.
3 detection of bacterial endotoxin of embodiment
4,10 × 75mm test tube (or reagents original ampoule of 0.1mL/ branch specification) equipped with 0.1mL reagents solution,
In 2 addition 0.1mL test samples as test product pipe, 12 λ endotoxin working standard 0.1mL of addition is as positive control pipe, 1
The reagents solution 0.1mL worn is added as negative control pipe in branch.After test tube is mixed gently, nozzle is closed, is vertically put into
In 37 ± 1 DEG C of water-baths, 60 ± 2 minutes are kept the temperature.Insulating process and test tube of taking are careful, and are avoided because of knot negative caused by being vibrated
Fruit.
As a result judge: test tube is gently taken out from water-bath, when slowly reversing 180 °, pipe inner gel is indeformable, not from pipe
Wall slippage person is that the positive is recorded as (+), and gel is not able to maintain complete and is feminine gender from tube wall slippage person, is recorded as (-).Test sample
2 pipes are for example (-), it is considered that meet regulation, no longer progress pyrogen test (Rabbit viscera) test.If 2 pipes are (+), it is considered that
It is against regulation.If 1 pipe is (+) in 2 pipes, 1 pipe is (-), separately takes 4 test sample pipe retrials according to the above method, has 1 pipe in 4 pipes
Think for (+) against regulation.
As a result: bacterial endotoxin reaches≤0.5EU/mg.
The detection of 4 systemic acute toxi-city of embodiment
Test method: selecting healthy mice 20, and for weight in 17-23g, male and female are each flat, is randomly divided into experimental group and right
According to group.Take leaching liquor that experimental group small white mouse is injected intraperitoneally, control group injecting normal saline, the upper and lower noon is each primary, injection
Dosage is 0.3mL/10g weight, continuous 7 days, observes its sign situation.
Test result: having no toxic reaction in acute toxicity test, small white mouse all survives, weight gain.Experimental group and
Control group weight no significant difference.Illustrate that collagen protein sponge used in this test will not cause acute poisoning symptom.
The detection of 5 cytotoxicity of embodiment
Test method: this experiment is using the leaching liquor of MTT colorimetric determination collagen protein sponge to fibroblast 2,4,7
The cytotoxicity of collagen protein sponge is evaluated in the influence of its opposite proliferation rate.Collagen protein sponge 1cm to be measured2It is thin that 10mL is added
Born of the same parents' culture solution sets 37 DEG C of standings and takes supernatant as the material leaching liquor of 100% concentration in 24 hours；Cell culture fluid multiple proportions is used again
Dilution, it is spare to be made 50% material leaching liquor.
The measurement of cell opposite proliferation rate and Materials Cell toxicity assessment: with the mouse skin of cell culture fluid logarithmic growth phase
It is 6 × 10 that fibroblast (2-3 generation), which is made into concentration,4A/mL cell suspension, every 100 μ L of hole, is inoculated in 96 well culture plates,
Set 37 DEG C, 5%CO2It is cultivated 24 hours in incubator；(200 μ L/ are replaced with the material leaching liquor of 50%, 100% concentration respectively
Hole), using cell culture fluid as blank control, continue to overturn away liquid in 2 holes after setting 37 DEG C of cultures 4 hours, every hole adds
DMSO150 μ L shakes 10min；Enzyme linked immunological instrument measures the absorbance value in every hole, and each group 8 take mean value.Measuring wavelength is
490nm.The cell opposite proliferation rate (RGR) of sample is calculated by formula R=experimental group OD/ control group OD × 100% later, and is pressed
The following table 1 evaluates the cytotoxicity classification of collagen protein sponge.
The cytotoxicity of 1 collagen protein sponge of table is classified
| RGR (%) | Cytotoxicity classification |
| ≥100 | 0 |
| 75-99 | I |
| 50-74 | II |
| 25-49 | III |
| 1-24 | IV |
| 0 | V |
Test result: the collagen protein sponge leaching liquor of concentration 50%, 100%, 2 days cell opposite proliferation rates may be up to
99% or more, 4 days, 7 days opposite proliferation rates extend declined at any time, but still 80% or more, cytotoxicity is divided into I
Grade.
Cytotoxicity reaches≤1 grade.
The test of 6 haemolysis of embodiment
Test method: rabbit heart blood 10-20mL is taken, it is that 2%RBC suspension is spare that volume fraction, which is made, in separating red corpuscle.
Test is divided into test product pipe, negative control pipe and positive control pipe, and every group sets 4 parallel pipes.Developmental tube is each concentration leaching liquor pair
The dilution of physiological saline, negative control use physiological saline, and positive control uses distilled water.The corresponding sample of 10mL is added in each pipe
Product after 37 DEG C of water-soluble 30min, respectively plus the RBC suspension of 0.2mL, are stood overnight, centrifuged supernatant exists after 37 DEG C of water-soluble 1h
Colorimetric at 520cm.
Test result: the hemolysis rate of experimental group and physiological saline group is respectively less than 5% in hemolytic test, distilled water group it is molten
Blood rate is 100%.Illustrate that testing collagen protein sponge used does not cause hemolytic reaction.
The test of 7 sensitization of skin of embodiment
Test method: taking healthy guinea pig 12, the next day be injected intraperitoneally 0.2mL leaching liquor, continuous 3 times, wherein 6 in for the first time
After injection continuous 14 days by be injected intraperitoneally 2mL leaching liquor；Other 6 21 days intraperitoneal injection 2mL leaching liquors after injecting for the first time.Often
There is useless grab to scratch nose, sneeze, perpendicular hair, twitch, expiratory dyspnea, size after observing 15 minutes, especially final injection after secondary injection
The reaction such as fecal incontinence, body temperature change, shock, death.
Test result: in sensitivity test after guinea pig intraperitoneal injection, whole cavys are survived, experimental group and control group cavy
Behavioral activity situation no significant difference.Illustrate that collagen protein sponge used in this test does not lead to body allergic reaction.
Sensitization rate reaches≤8%.
The intradermal stimulation of embodiment 8 test
Test method: rabbit quadriceps muscle of thigh test injection: new zealand rabbit 10, right hindlimb quadriceps muscle of thigh is test area,
1-2mL leaching liquor is injected, left side corresponding position is injected isometric physiological saline with method and compared, and 48 hours, 14 days, 21 are injected
Test rabbit is put to death in its grouping, and quadriceps muscle of thigh is taken out in dissection, longitudinally slit, observation injection site musculature variation: (one) grade, yin
Property reaction, it is nonirritant, medicine-feeding part with compare position indifference；(+) grade, suspicious reaction, medicine-feeding part musculature is congested,
Diameter is in 0.5cm or so；(++) grade, mild reaction, medicine-feeding part musculature is congested, and diameter is in 1cm or so；(+++) grade, weight
Degree reaction, medicine-feeding part tissue is red and swollen, blue, and gloss disappears, it is seen that downright bad point.Tissue send pathologic finding simultaneously.
Test result: quadriceps muscle of thigh injects leaching liquor and physiological saline 48 hours, 14 days, 21 days, and each experimental group naked eyes are seen
Tissue is examined without significant reaction；Swelling between tissue striated muscle is seen by tissue pathology checking within 48 hours, but has no apparent inflammatory reaction；
See that rhabdium swelling degree gradually mitigates within 14th day and the 21st day, has no apparent inflammatory reaction, control group and experimental group
The extent of reaction is identical.Test result illustrates that collagen protein sponge used in this experiment has no stimulation to deep tissue, does not generate bright
Aobvious tissue reaction.
9 Implantation Test of embodiment
Test method: male mouse of kunming 28, weight 25-30g.Under aseptic condition, collagen protein sponge is punched
The former piece of diameter 1cm is made in device；Fixed after mouse anesthesia, the disinfection of back center, unhairing, the stringer for cutting off about 0.7cm or so are cut
Mouthful, collagen protein sponge is put into right side subcutaneously, it is normal to raise after 2 needle of catgut suture.Postoperative 3,5,7,9 days optional 7 of difference
It visually observes and draws materials after execution and carry out routine histologic inspection.
Test result: gross examination of skeletal muscle: postoperative 3 day, it is closer to be shown in that collagen protein sponge is attached with substrate, is not easy to uncover, but
It can completely take out, substantially without inflammatory reaction, surrounding materials tissue is without necrosis, abscess；5 days after operation, collagen protein sponge have dropped
Solve thinning, surface is wrapped up by a few fibres film, and tunica fibrosa is wrapped up on removal surface, and material is complete, and surface is smooth；Postoperative 7 days, collagen
Protein sponge it is obvious it is thinning, become smaller, material is micro- in faint yellow, and surface is smooth；Postoperative 9 days, material was substantially completely degraded, on a small quantity
Residual fraction has been not easy completely to separate.Histomorphological: postoperative 3 days, it is seen that have a little neutral grain in collagen protein sponge
Inflammatory cell infiltration based on cell, lymphocyte, collagen protein sponge reticular structure are clear；Postoperative 9 days, collagen protein sponge
It fully absorbs, is replaced fibrous connective tissue.Each time has no tissue degeneratiaon, necrosis.
After muscular grafting 7 days, the inflammatory cell extent of reaction≤IV grades；After implantation 15 days, the inflammatory cell extent of reaction≤III
Grade；Implantation 30 days after, the inflammatory cell extent of reaction≤II grade, and around be no different paradoxical reaction 6 weeks after this product be absorbed.
The test of 10 genetoxic of embodiment
Gene mutation is detected by Salmonella reversion test；
Chromosome aberration is detected by micronucleus test；
It is distorted by chromosome aberrations in spermatgognium testing inspection genome；
Pass through single cell gel electrophoresis analysis detection DNA initial injury.
Test result: hereditary-less toxicity.
The degradation test of embodiment 11
Enzymatic isolation method, starch is placed in the physiological saline containing a- amylase and carbohydrase at a temperature of 37 DEG C ± 2 DEG C in product
In after 72 hours, under a- amylase and saccharification enzyme effect, be converted into glucose, conversion ratio is equal to degradation rate.
Through examining, product is subcutaneously implanted 6 weeks and is degraded and absorbed, and histologic reaction is good.
Product does not discharge the substance that any pair of place patient generates side effect, meets the biology that GB/T16886.1 is provided and comments
Valence guide.Collagen protein sponge provided by the invention has good biocompatibility, the nontoxic secondary work of inside and outside safety experiment
With.
The invention is not limited to above embodiment, if not departing from the present invention to various changes or modifications of the invention
Spirit and scope, if these modification and variations belong within the scope of claim and equivalent technologies of the invention, then this hair
It is bright to be also intended to encompass these changes and change.
Claims (10)
1. a kind of biocompatibility detection method of collagen protein sponge, which comprises the following steps:
(1) preparation of collagen protein sponge leaching liquor；
(2) Sterility testing of collagen protein sponge；
(3) detection of bacterial endotoxin of collagen protein sponge；
(4) the systemic acute toxi-city detection of collagen protein sponge；
(5) the cytotoxicity detection of collagen protein sponge；
(6) the hemolytic test of collagen protein sponge；
(7) the hypersensitive test of collagen protein sponge；
(8) picosecond laser pulse of collagen protein sponge；
(9) Implantation Test of collagen protein sponge；
(10) genetic toxicity test of collagen protein sponge；
(11) the degradation detection of collagen protein sponge.
2. biocompatibility detection method according to claim 1, it is characterised in that: in step (1), the leaching liquor
Preparation are as follows: the proportion that collagen protein sponge is pressed to 0.5-2cm/mL is impregnated 24-48 hours with physiological saline.
3. biocompatibility detection method according to claim 1, it is characterised in that: in step (4), the whole body is acute
Toxicity detection are as follows: select healthy mice even numbers only, for weight in 17-23g, male and female are each flat, and mean random is divided into experimental group and right
According to group, take leaching liquor that experimental group small white mouse is injected intraperitoneally, control group injecting normal saline, the upper and lower noon is each primary, injection
Dosage is 0.2-0.6mL/10g weight, continuous 7 days, observes its sign situation.
4. biocompatibility detection method according to claim 1, it is characterised in that: in step (5), the cytotoxicity
Detection are as follows: using the leaching liquor of MTT colorimetric determination collagen protein sponge to the shadows of 2,4,7 days opposite proliferation rates of fibroblast
It rings, evaluates the cytotoxicity of collagen protein sponge.
5. biocompatibility detection method according to claim 1, it is characterised in that: in step (6), the hemolytic examination
It tests are as follows: take rabbit heart blood 10-20mL, it is spare that the RBC suspension that volume fraction is 2% is made in separating red corpuscle；Test is divided into examination
Quality control, negative control pipe and positive control pipe, every group sets parallel pipe；Developmental tube is dilution of each concentration leaching liquor to physiological saline
Liquid, negative control use physiological saline, and positive control uses distilled water；8-20mL respective sample, 37 DEG C of water-soluble 30- are added in each pipe
After 60min, respectively plus the RBC suspension of 0.16-0.4mL, stood overnight after 37 DEG C of water-soluble 1-2h, centrifuged supernatant exists
Colorimetric at 520cm.
6. biocompatibility detection method according to claim 1, it is characterised in that: in step (7), the sensitization of skin
Property test are as follows: take healthy guinea pig even numbers only, the next day be injected intraperitoneally 0.15-0.5mL leaching liquor, continuous 3 times, wherein half cavy in
For the first time inject after continuous 14 days by be injected intraperitoneally 1.5-2.5mL leaching liquor；In addition half cavy 21 days abdominal cavities after injecting for the first time
Inject 1.5-2.5mL leaching liquor；There is useless grab scratch nose, sneeze, erect after observing 15 minutes, especially final injection after per injection
The reaction such as hair, twitch, the change of expiratory dyspnea, gatism, body temperature, shock, death.
7. biocompatibility detection method according to claim 1, it is characterised in that: in step (8), the intradermal stimulation
Test are as follows: using rabbit as object, right hindlimb quadriceps muscle of thigh is test area, injects 1-2mL leaching liquor, and left side corresponding position is same
Method is injected isometric physiological saline and is compared, and 48 hours, 14 days, the execution test rabbit of grouping in 21 days are injected, and stock four is taken out in dissection
Head flesh, longitudinally slit, observation injection site musculature variation.
8. biocompatibility detection method according to claim 1, it is characterised in that: in step (9), the Implantation Test
Are as follows: take male mouse of kunming, weight 25-30g；Under aseptic condition, collagen protein sponge is made to the former piece of diameter 0.5-1cm；
Fixed after mouse anesthesia, the disinfection of back center, unhairing cut off the stringer notch of about 0.7cm or so, collagen protein sponge are put into
Right side is subcutaneous, normal to raise after catgut suture；Postoperative 3,5,7,9 natural gift other places, which after death visually observe and draw materials, carries out conventional group
It knits to check.
9. biocompatibility detection method according to claim 1, it is characterised in that: in step (11), the degradation inspection
It surveys are as follows: it is small to be placed in 72-96 in the physiological saline containing a- amylase and carbohydrase at a temperature of 37 DEG C ± 2 DEG C for starch in product
Shi Hou is converted into glucose under a- amylase and saccharification enzyme effect, and conversion ratio is equal to degradation rate.
10. biocompatibility detection method according to claim 1, which is characterized in that the biocompatibility detection is answered
When meeting following index:
The collagen protein sponge is sterile after Co-60 ray sterilizing；
The bacterial endotoxin reaches≤0.5EU/mg；
The collagen protein sponge will not cause acute poisoning symptom；
The cytotoxicity reaches≤1 grade；
The collagen protein sponge does not cause hemolytic reaction；
The collagen protein sponge does not lead to body allergic reaction；
The collagen protein sponge has no stimulation to deep tissue, has no apparent inflammatory reaction, does not generate apparent tissue
Reaction；
The collagen protein sponge is after muscular grafting 7 days, the inflammatory cell extent of reaction≤IV grades；After implantation 15 days, inflammation is thin
Born of the same parents' extent of reaction≤III level；After implantation 30 days, the inflammatory cell extent of reaction≤II grades, and inhaled after being around no different paradoxical reaction 6 weeks
It receives；
The collagen protein sponge hereditary-less toxicity.
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| CN114397293B (en) * | 2021-12-30 | 2024-05-14 | 卓阮医疗科技（苏州）有限公司 | Collagen-based biological material endotoxin extraction and detection method |
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| CN114397293A (en) * | 2021-12-30 | 2022-04-26 | 卓阮医疗科技（苏州）有限公司 | Extraction and detection method for endotoxin in collagen-based biological material |
| CN114397293B (en) * | 2021-12-30 | 2024-05-14 | 卓阮医疗科技（苏州）有限公司 | Collagen-based biological material endotoxin extraction and detection method |
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Application publication date: 20181211 |