CN108853571A - A kind of collagen protein sponge and its preparation process - Google Patents
A kind of collagen protein sponge and its preparation process Download PDFInfo
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- CN108853571A CN108853571A CN201810813851.6A CN201810813851A CN108853571A CN 108853571 A CN108853571 A CN 108853571A CN 201810813851 A CN201810813851 A CN 201810813851A CN 108853571 A CN108853571 A CN 108853571A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/001—Use of materials characterised by their function or physical properties
- A61L24/0036—Porous materials, e.g. foams or sponges
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/001—Use of materials characterised by their function or physical properties
- A61L24/0042—Materials resorbable by the body
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
- A61L24/10—Polypeptides; Proteins
- A61L24/102—Collagen
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/04—Materials for stopping bleeding
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Abstract
The invention discloses a kind of collagen protein sponge and its preparation process, preparation process includes the following steps：(1) collagen raw material carry out pre-treatment；(2) the preceding extraction of collagen raw material；(3) enzymatic hydrolysis, low-temperature centrifugation, purifying of saltouing, dialysis, vacuum homogeneous obtain collagen；(4) collagen protein sponge is prepared；(5) dress, sterilizing, outer packing is wrapped inside to get product collagen protein sponge in collagen protein sponge.It supplies disposable collagen haemostatic sponge with germ-free condition, the product has good physicochemical property and biology performance, can be degraded and absorbed, advantageously promote wound healing, suitable for capillary, vein and artery hemostasis and diffusivity bleeding.
Description
Technical field
The present invention relates to a kind of sponge and its preparation processes, belong to pharmaceutical technology field, in particular to a kind of with excellent
Biocompatibility collagen protein sponge and its preparation process.
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.
Currently used hemostatic material such as gelfoam, regenerated fiber hemostatic gauze etc. has been applied for many years in clinic, but not
Same material has the advantages that different hemostatic mechanism and corresponding drawn game limit, especially pure Animal by-product wind with higher
Danger.Now most sponges are gelfoam on the market, and gelatin extracts from leather more, and triple helix structure has been destroyed, average molecular
Mass Distribution is wider, and it is uncontrollable not have bioactivity, material source, has been easy heavy-metal residual, and has led to trauma surface infestation.
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 collagen protein sponge and its preparation processes.
The product is to be suitable for germ-free condition supply and disposable styptic sponge to capillary, vein and artery hemostasis,
And diffusivity bleeding.
In order to achieve the above object, the present invention uses following technical scheme：
The present invention provides a kind of preparation process of collagen protein sponge, includes the following steps：
(1) collagen raw material carry out pre-treatment；
(2) the preceding extraction of collagen raw material；
(3) enzymatic hydrolysis, low-temperature centrifugation, purifying of saltouing, dialysis, vacuum homogeneous obtain collagen；
(4) collagen protein sponge is prepared；
(5) dress, sterilizing, outer packing is wrapped inside in collagen protein sponge.
Further, in step (1), the pre-treatment is：By fascia, the rouge in the heel string through examining qualified, healthy ox
Fat, muscle pick, and are rinsed well with tap water, are cut into the thin slice that thickness is no more than 1mm.
Further, it in step (2), is extracted as before described：By the collagen raw material after pre-treatment be put into 7-12 DEG C it is pure
Change and impregnated in water, impregnates no less than 16 hours, rinsed to solution with purified water in clear state.
Further, in step (3), the enzymolysis liquid allocation ratio of the enzymatic hydrolysis is：750-800L purified water, adds 28-32L
Glacial acetic acid controls pH value in 2.5-3.5 after adding 50-55g pepsin to be sufficiently stirred；
The enzymolysis step is：By in the collagen raw material extracted before menstruation and enzymolysis liquid investment enzymatic vessel, sufficiently stir
It mixes, enzymolysis time is no less than 72 hours.
Further, in step (3), the low-temperature centrifugation is：4 DEG C, 3000 revs/min, centrifugation time is 20 minutes；It collects
Supernatant.
Further, in step (3), the purifying of saltouing is：Supernatant obtained by low-temperature centrifugation is added in NaCl solution,
White flock collagen is precipitated；The NaCl solution needs the filter screen filtration of 360 mesh, and strainer uses after should sterilizing, to remove
Decontamination improves the purity of NaCl solution.
Further, in step (3), the dialyzate of the dialysis is phosphate buffer, and dialyzate pH value is in 2.5-3.5
In range；
The dialysis step is：By bag filter cut into 350mm long and with purified water foam washing it is clean, then with boiling water boiling 2
Hour, the collagen for purifying of saltouing is fitted into the bag filter for the molecular weight 12000-14000 that shuts off and is dialysed；
The requirement that dialyzate changes liquid is：Out of 1-6 day, pH value is in 2.5-3.5, replacement in every 3 days dialyzate 1 time；From
In 7-11 days, pH value is in 3.5-4.5, daily replacement dialyzate 1 time；From the 12nd day, pH value in 4.5-5.5, according to
It needs to change liquid.
Further, in step (3), the vacuum homogeneous is：By the collagen after dialysis within the scope of pH value 5.0-5.5
It is put into vacuum viscolizer, revolution is controlled at 70-80 revs/min, and vacuum degree is set in -0.9Pa, vacuum to bubble-free.
Further, in step (4), it is described prepare collagen protein sponge the specific steps are：By the collagen egg after homogeneous
It is white to be fitted into sterilized lyophilized plate, then lyophilized plate is fitted into drying box of freeze dryer；Product from room temperature be chilled to -40 DEG C with
Under, heat preservation no less than 4 hours, into vacuum operation, it is heated to 30-40 DEG C after being warming up to 5 DEG C naturally under vacuum state, is made
Product is at platinum sponge sheet.
The present invention provides a kind of collagen protein sponge, is made by preparation process as described above.
Compared with prior art, the invention has the advantages that：
(1) present invention avoids gelfoam product with germ-free condition supply and disposable collagen protein sponge
Have not have bioactivity, a material source uncontrollable, be easy heavy-metal residual, and led to the risk of trauma surface infestation；It should
Product can be degraded and absorbed, and histologic reaction is good, can advantageously promote wound healing；
(2) product collagen protein sponge of the invention has good physicochemical property and biology performance, does not discharge and takes the post as
What generates the substance of side effect to patient, has good biocompatibility, and inside and outside safety experiment has no toxic side effect；Through trying
It tests, is valid up to 2 years；
(3) product collagen protein sponge of the invention is suitable for capillary, vein and artery hemostasis and diffusivity
Bleeding can be widely used in various wounds and the hemostasis of all kinds of surgical wound surfaces.
Detailed description of the invention
Fig. 1 is collagen protein sponge production technological process；
Fig. 2 a and Fig. 2 b are respectively the scanning electron microscope sectional view and plane of collagen protein sponge in compliance test result embodiment 2
Figure.
Specific embodiment
Now in conjunction with attached drawing and specific embodiment, invention is further described in detail for effect experimental examples.
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.
Collagen protein sponge of the present invention production include the selection of organization material, pretreatment, collagen extraction and purification, be detailed in
Process flow chart, i.e., shown in Fig. 1.
Product after being packaged sterilizing should be stored in relative humidity no more than 65%, non-corrosive gas, cool place, drying,
Draughty interior.
Embodiment 1
1.1 raw material
Through the heel string for examining qualified, healthy ox.
1.1.1 it slightly washes
Between guaranteeing slightly to wash, under the premise of vessel, cutter are clean, fascia, fat, muscle are picked first, use tap water
It rinses well.
1.1.2 fine purifiation
Slightly washed ox heel string is rinsed with purified water between fine purifiation, mounted box freezing is stand-by.
1.1.3 raw material crushes
With alcohol wipe slicer, sterilizing rear be can be used.Ox heel string is cut into the thin slice that thickness is no more than 1mm.
It is extracted before 1.2
Raw material after slice is put into 7-12 DEG C of purified water and is impregnated, impregnate no less than 16 hours, with purified water rinse to
Solution is in clear state.
1.3 enzymatic hydrolysis
Enzymatic vessel is sterile-processed to be used after the assay was approved, by enzymatic vessel with purified water to impregnating in tank, after immersion
Top tank structure is rinsed with purified water to be no less than 3 times, until after range estimation noresidue, then top tank structure and fan are slowly rinsed with glacial acetic acid
Leaf, glacial acetic acid attachment time are no less than 30 minutes.Top tank structure is rinsed with purified water to be no less than 3 times, until after top tank structure is without tart flavour,
It is artificial to clean top tank structure and flabellum with clean rag, it cleans number and is no less than 3 times, then rinsed 3 times with purified water, until flushing water
Clear, then with agitating device in 75% alcohol rinse tank inner surface and tank, continuous flushing 3 times from top to bottom, then with purifying
Water rinses 6 times, and agitating device, each blade are intended to rinse 6 times in tank, and inspector should sample in time carries out microbial limit inspection
It looks into.
After passing the inspection, operator should prepare enzymolysis liquid under the supervision of inspector, and the allocation ratio of enzymolysis liquid is：
750-800L purified water adds 28-32L glacial acetic acid, after adding 50-55g pepsin to be sufficiently stirred control pH value 2.5-3.5 i.e.
Can, the ox heel string 5.5-6.5g and enzymolysis liquid that extract before menstruation are put into enzymatic vessel, are sufficiently stirred, it is small that enzymolysis time is no less than 72
When.
1.4 low-temperature centrifugation
Every glass of 1L of Centrifuge Cup, 6 glasss of need balance, and each balance error is no more than ± 5g.
Centrifuge temperature is set as 4 DEG C, and running speed is 3000 revs/min, and centrifugation time is 20 minutes.
It is conscientious to need when collecting supernatant, must not pour into decorating film in supernatant.
1.5 saltout purifying
NaCl solution needs the filter screen filtration of 360 mesh, and strainer uses after should sterilizing, and to remove impurity, it is molten to improve NaCl
The purity of liquid.
Supernatant is added in NaCl solution, white flock collagen is precipitated.
1.6 dialysis
Dialysate tank is sterile-processed to be used after the assay was approved, and bag filter uses after being sterilized.Dialysate tank purified water pair
It is impregnated in tank, rinses top tank structure with purified water after immersion and be no less than 3 times, until after range estimation noresidue, then use glacial acetic acid
Top tank structure and flabellum are slowly rinsed, the glacial acetic acid attachment time is no less than 30 minutes.Top tank structure is rinsed with purified water to be no less than 3 times,
Until after top tank structure is without tart flavour, it is artificial to clean top tank structure and flabellum with clean rag, it cleans number and is no less than 3 times, then with pure
Change water to rinse 3 times, until flushing water clear, then with agitating device in 75% alcohol rinse tank inner surface and tank, from top to bottom
Continuous flushing 3 times, then rinsed 6 times with purified water, agitating device, each blade are intended to rinse 6 times in tank, and inspector should take in time
Sample carries out limit test of microbe, prepares dialyzate phosphate buffer under inspector's supervision.
By bag filter cut into 350mm long and with purified water foam washing it is clean, then with stand-by after boiling water boiling 2 hours.
The collagen for purifying of saltouing is fitted into the bag filter for the molecular weight 12000-14000 that shuts off and is dialysed.It prepares for the first time
Dialyzate pH value is within the scope of 2.5-3.5.
It is recorded by lot number, lot number board is used dialysate tank to identify respectively.
Dialyzate changes the requirement of liquid：
(1) out of 1-6 day, pH value is in 2.5-3.5, replacement in every 3 days dialyzate 1 time
(2) out of 7-11 day, pH value is in 3.5-4.5, daily replacement dialyzate 1 time.
(3) from the 12nd day, pH value changes liquid in 4.5-5.5 as needed.
1.7 vacuum homogeneous
Homogeneous tank every batch of before homogeneous, needs sterile-processed rear use for the first time.By homogeneous tank to being impregnated in tank, after immersion
Top tank structure is rinsed with purified water to be no less than 3 times, until after range estimation noresidue, then top tank structure and fan are slowly rinsed with glacial acetic acid
Leaf, glacial acetic acid attachment time are no less than 30 minutes.Top tank structure is rinsed with purified water to be no less than 3 times, until after top tank structure is without tart flavour,
It is artificial to clean top tank structure and flabellum with clean rag, it cleans number and is no less than 3 times, then rinsed 3 times with purified water, until flushing water
Clear, then with agitating device in 75% alcohol rinse tank inner surface and tank, continuous flushing 3 times from top to bottom, then with purifying
Water rinses 6 times, and agitating device, each blade are intended to rinse 6 times in tank, and inspector should sample in time carries out microbial limit inspection
It looks into.
After passing the inspection, the collagen after dialysis within the scope of pH value 5.0-5.5 is put into vacuum viscolizer, revolution
At 70-80 revs/min, vacuum degree is set in -0.9Pa, vacuum to bubble-free for control.
1.8 filling
Collagen after homogeneous is fitted into sterilized lyophilized plate, lyophilized plate is then packed into drying box of freeze dryer
In.
1.9 freeze-drying
Product is chilled to -40 DEG C from room temperature and is no less than 4 hours hereinafter, keeping the temperature, natural under vacuum state into vacuum operation
It is heated to 30-40 DEG C after being warming up to 5 DEG C, makes product at platinum sponge sheet.
1.10 inner packing
Freeze-dried products are taken out from drying box of freeze dryer, specification as required is cut, and installation verification lot number is errorless, will be wrapped
After table top of installing is sterilized with 75% alcohol wipe, product inner packing is carried out.
Packing machine should all check the upper heating of setting, lower heating, whether seal temperature is set in adds before each packaging
Heat is 80 DEG C ± 5 DEG C, heat-sealing is at 140 DEG C ± 5 DEG C.
Inner packing answers continuous stamping to be no less than 20 empty packages before carrying out product packaging, carries out product packet again after inspection
Dress.
Whether accurate, sealing machine temperature is set in 220 DEG C to verification compound membrane bag stamp, and speed is set as 6 times/min, will be complete
It is packed into compound membrane bag and seals at the product of one layer of inner packing.
1.11 sterilizing
Turnover box is added by GB18280-2000idt ISO 11137 in the interior product wrapped:1995《Healthcare products go out
Bacterium confirmation and conventional control require radiation sterilization》Select 25kGy dosage.
1.12 outer packing
It will sterilize via radiation and examine qualified product to be fitted into the middle packing box for accomplishing fluently lot number together with specification and be put into
Middle packaging is finally packed into big packing case by the quality certification, the good product batch number of big packing case outer cover and sterilization date etc..
Compliance test result embodiment 1
Biocompatibility is the extremely important performance of biomedical material, to ensure safety of the materials'use after human body
Property, according to, to the biocompatibility test project demand of collagen product, reference Qi Chen edits in GB/T16886.1-2001
《Herbal pharmacology experimental method》Beijing：People's Health Publisher, 1994.46-108 test methods provided, our company is produced
Collagen protein sponge impregnated 24 hours with physiological saline, extracting solution taken to carry out acute poison by the proportion of 1.25cm/mL
Property test, irritation test, sensitivity test and hemolytic test, cell toxicity test and carried out skin with collagen protein sponge
Lower Implantation Test.
2.1. sterile
According to《Pharmacopoeia of People's Republic of China》The regulation of 2010 editions two annex XI H is detected, and rule are as a result met
It is fixed：
Product is sterile after Co-60 ray sterilizing.
2.2. bacterial endotoxin
According to《Pharmacopoeia of People's Republic of China》The two annex XI E methods of version in 2010 are detected, and rule are as a result met
It is fixed：
Bacterial endotoxin reaches≤0.5EU/mg.
2.3. systemic acute toxi-city
Leaching liquor is taken to be detected by GB/T16886.11：
Test method：Healthy mice 20 are selected, for weight in 17-23g, male and female are each flat, are 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：Toxic reaction is had no 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.
2.4. cytotoxicity
It takes leaching liquor to be detected by leaching liquor method in GB/T16886.5, as a result meets regulation：
Cell toxicity test is the screening test of first stage in medical material Evaluation of Biocompatibility.Nineteen eighty-three, by Mos
Man reports a kind of Fast Evaluation cell Proliferation for the first time and the colorimetric analysis (MTT colorimetric method) of cytotoxicity is wide at present
The general evaluation of its biocompatibility applied to medical material.
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 calculates the cell opposite proliferation rate (RGR) of sample by formula R=experimental group OD/ control group OD × 100% later and presses
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.
2.5. haemolysis
It is detected by contact method in GB/T16886.4, hemolysis rate should be less than 5%, as a result meet regulation：
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.
2.6. sensitization of skin
It is detected by maximum dose method in GB/T16886.10：
Test method：Take 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%.
2.7. intradermal stimulation
It takes leaching liquor to be detected by GB/T16886.10, meets regulation：
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：(1) 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.
2.8. Implantation Test
It is tested according to test method as defined in GB/T16886.6：
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 days, it is closer to see 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；After implantation 30 days, the inflammatory cell extent of reaction≤II grades, and surrounding is no different paradoxical reaction, this product is absorbed after 6 weeks.
2.9. genetoxic
It is tested according to test method as defined in GB/T16886.3, as a result meets regulation：
Hereditary-less toxicity.
2.10. degradation
Enzymatic isolation method is examined by GB/T16287-1996 method, in product starch at a temperature of 37 DEG C ± 2 DEG C, be placed in containing
In the physiological saline of a- amylase and carbohydrase after 72 hours, under a- amylase and saccharification enzyme effect, it is 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 release the substance that any couple of 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.
Compliance test result embodiment 2
The physicochemical property of product and the stability of biological efficiency are to determine the important symbol of keeping life.This product ginseng
It according to same kind of products at abroad and the provisions of the relevant regulations issued by the State, will fix tentatively 2 years validity period, while same batch products will be carried out 4 years by a definite date
Real-time tracing research, research contents is as follows：
3.1. Testing index
3.1.1. the physical property detection of collagen protein sponge
3.1.1.1. the appearance of collagen protein sponge and surface texture (annual periodically primary)：
The appearance of collagen protein sponge under natural light by visually observing.Surface texture is seen by scanning electron microscope (SEM)
It examines, is completed by central laboratory of Yangzhou University.
3.1.1.2. water absorbing capacity (every half a year periodically carries out once)：
Collagen protein sponge 5 are taken, electronic balance precision weighs every tablet quality.It immerses in 20mL room temperature (25-30 DEG C) water,
After suctioning moisture, one jiao gently being clamped with tweezers, is left the water after draining 1min, electronic balance weighing obtains every water suction
Quality afterwards.Water absorbing capacity is calculated by method：
Quality/sample quality after water absorbing capacity (again)=water suction
3.1.1.3. mass per volume (apparent density) (every half a year periodically carries out once)：
Collagen protein sponge 5 are taken, length, width and the height of accurate measurement every calculate sample volume；Electronics day
It is flat to weigh quality, the quality in every sponge unit volume is calculated, mean value is taken.
3.1.2. the chemical property detection (annual periodically to carry out once) of collagen protein sponge
3.1.2.1.pH value measurement
Take collagen protein sponge 5, every tear after be immersed in 30min in 15mL distilled water, according to《Chinese people's republicanism
State's pharmacopeia》The measurement of method as defined in two annex.
3.1.2.2. total protein, proline and hydroxyproline content measure
Total protein content measurement measures examination using the biuret method total protein content of Shanghai Rongsheng Bioisystech Co., Ltd
Agent box, standard protein use the collagen of Sigma company, are dissolved after accurately weighing with spirit of vinegar.Collagen protein sponge sample
It is dissolved with 20mL spirit of vinegar, lysate is taken to measure.
Calculation method：
Total protein content=total protein measured value/collagen protein sponge quality × 100%
Hydroxyproline content measurement uses chloramine-t method.The hydroxyproline determination of Bioengineering Research Institute is built up using Nanjing
Kit.6N HCl tube sealing is added after collagen protein sponge sample precise, 110 DEG C of hydrolysis take hydrolyzate to measure.
Calculation method：
Hydroxyproline content=hydroxyproline determination value/total protein measured value × 100%
3.1.3. collagen protein sponge biological property detects
3.1.3.1. liver hemostasis and in vivo Implant experiment (each year periodically carries out one-time detection)
New zealand white rabbit 15, weight 2-2.5kg are taken, is organized at two in same lobectomy of liver liver 1cm, oozing of blood is formed
The surface of a wound uses collagen protein sponge and gelfoam (production of Nanjing third pharmaceutical factory) apply pressure surface of a wound respectively, records bleeding stopping period；Only
It is sutured after blood at four angles of sponge and the liver surface of a wound, its absorption degradation situation of routine observation simultaneously carries out pathological examination.
3.1.3.2. collagen protein sponge Sterility testing (annual periodically to carry out one-time detection)
Testing result：
The testing result of collagen protein sponge physical property is as shown in table 2 below：
The testing result of 2 collagen protein sponge physical property of table
The testing result of collagen protein sponge chemical property is as shown in table 3 below：
The testing result of 3 collagen protein sponge chemical property of table
The testing result of collagen protein sponge biological property is as shown in table 4 below：
The testing result of 4 collagen protein sponge biological property of table
| Detection time | Testing index | Testing result |
| 2014.12 | It answers sterile | It is sterile |
| 2015.12 | It answers sterile | It is sterile |
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 preparation process of collagen protein sponge, which is characterized in that include the following steps：
(1) collagen raw material carry out pre-treatment；
(2) the preceding extraction of collagen raw material；
(3) enzymatic hydrolysis, low-temperature centrifugation, purifying of saltouing, dialysis, vacuum homogeneous obtain collagen；
(4) collagen protein sponge is prepared；
(5) dress, sterilizing, outer packing is wrapped inside in collagen protein sponge.
2. preparation process according to claim 1, it is characterised in that：In step (1), the pre-treatment is：It will be through examining
Fascia, fat, muscle in the heel string of qualified, healthy ox pick, and are rinsed well with tap water, are cut into thickness no more than 1mm's
Thin slice.
3. preparation process according to claim 1, it is characterised in that：In step (2), it is extracted as before described：By pre-treatment
Collagen raw material afterwards, which is put into 7-12 DEG C of purified water, to be impregnated, and is impregnated no less than 16 hours, is rinsed with purified water to solution and be in
Clear state.
4. preparation process according to claim 1, it is characterised in that：In step (3), the enzymolysis liquid of the enzymatic hydrolysis configures ratio
Example be：750-800L purified water adds 28-32L glacial acetic acid, and pH value is controlled after adding 50-55g pepsin to be sufficiently stirred in 2.5-
3.5。
5. preparation process according to claim 1, it is characterised in that：In step (3), the enzymolysis step is：It will be premenstrual
In collagen raw material and enzymolysis liquid the investment enzymatic vessel of extraction, it is sufficiently stirred, enzymolysis time is no less than 72 hours.
6. preparation process according to claim 1, it is characterised in that：In step (3), the dialyzate of the dialysis is phosphoric acid
Salt buffer, dialyzate pH value is within the scope of 2.5-3.5.
7. preparation process according to claim 1, it is characterised in that：In step (3), the dialysis step is：It will dialysis
Bag cut into 350mm long and clean with purified water foam washing, then with boiling water boiling 2 hours, by the collagen loading for purifying of saltouing
Shut off molecular weight 12000-14000 bag filter in dialyse；
The requirement that dialyzate changes liquid is：Out of 1-6 day, pH value is in 2.5-3.5, replacement in every 3 days dialyzate 1 time；From 7-
In 11 days, pH value is in 3.5-4.5, daily replacement dialyzate 1 time；From the 12nd day, pH value was in 4.5-5.5, as needed
Change liquid.
8. preparation process according to claim 1, it is characterised in that：In step (3),
The low-temperature centrifugation is：4 DEG C, 3000 revs/min, centrifugation time is 20 minutes；Collect supernatant；
The purifying of saltouing is：Supernatant obtained by low-temperature centrifugation is added in NaCl solution, white flock collagen is precipitated；Institute
The filter screen filtration that NaCl solution needs 360 mesh is stated, strainer uses after should sterilizing, and to remove impurity, improves the pure of NaCl solution
Degree；
The vacuum homogeneous is：Collagen after dialysis within the scope of pH value 5.0-5.5 is put into vacuum viscolizer, revolution control
For system at 70-80 revs/min, vacuum degree is set in -0.9Pa, vacuum to bubble-free.
9. preparation process according to claim 1, it is characterised in that：It is described to prepare collagen protein sponge tool in step (4)
Body step is：Collagen after homogeneous is fitted into sterilized lyophilized plate, it is dry that lyophilized plate is then packed into freeze dryer
In case；Product is chilled to -40 DEG C from room temperature and is no less than 4 hours hereinafter, keeping the temperature, and into vacuum operation, rises naturally under vacuum state
Temperature makes product at platinum sponge sheet to being heated to 30-40 DEG C after 5 DEG C.
10. a kind of collagen protein sponge, it is characterised in that：It is made by the described in any item preparation processes of claim 1-9.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109749116A (en) * | 2019-01-17 | 2019-05-14 | 杭州蓝朗生物技术有限公司 | A kind of foam type biomaterial membrane preparation method |
| CN113061283A (en) * | 2021-03-31 | 2021-07-02 | 广州创尔生物技术股份有限公司 | Method for preparing collagen sponge by using high-concentration collagen liquid |
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| CN105343928A (en) * | 2015-11-30 | 2016-02-24 | 江南大学 | Technology for preparing collagen sponge |
| CN106946988A (en) * | 2017-04-07 | 2017-07-14 | 李毅 | A kind of extracting method of ox heel string collagen |
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| CN1210019A (en) * | 1998-09-10 | 1999-03-10 | 战丽芬 | Medical collagen sponge and manufacture thereof |
| CN104628844A (en) * | 2013-11-11 | 2015-05-20 | 惠合再生医学生技股份有限公司 | Soluble collagen and preparation method thereof |
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| CN105343928A (en) * | 2015-11-30 | 2016-02-24 | 江南大学 | Technology for preparing collagen sponge |
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| CN113061283A (en) * | 2021-03-31 | 2021-07-02 | 广州创尔生物技术股份有限公司 | Method for preparing collagen sponge by using high-concentration collagen liquid |
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