CN108904861A - Collagen protein sponge promotes application and its physicochemical property detection method in wound healing drug in preparation - Google Patents
Collagen protein sponge promotes application and its physicochemical property detection method in wound healing drug in preparation Download PDFInfo
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- CN108904861A CN108904861A CN201810812939.6A CN201810812939A CN108904861A CN 108904861 A CN108904861 A CN 108904861A CN 201810812939 A CN201810812939 A CN 201810812939A CN 108904861 A CN108904861 A CN 108904861A
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- collagen
- sponge
- collagen protein
- water
- protein sponge
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Abstract
The present invention provides application and its physicochemical property detection method of the collagen protein sponge in preparation promotion wound healing drug.The collagen protein sponge can promote cell growth, differentiation, it can promote connective tissue reparation, with effectively hemoglutination, it can promote vascularization, accelerate granulation tissue growth, nutriment abundant is provided for the growth of tissue, the time for the surface of a wound that can heal in advance, accelerates the healing of injury tissue.The physicochemical property detection method, includes the following steps：(1) the physical property detection of collagen protein sponge, appearance and surface texture observation including collagen protein sponge, water absorbing capacity, the detection of mass per volume；(2) the chemical property detection of collagen protein sponge, including pH value measurement, total protein and hydroxyproline content measure, the measurement of residue on ignition, arsenic content, content of beary metal, pH value, readily oxidizable substance.
Description
Technical field
The present invention relates to a kind of sponges to promote application and its physicochemical property detection method in wound healing drug in preparation,
Belong to pharmaceutical technology field, in particular to a kind of collagen protein sponge promotes application and its reason in wound healing drug in preparation
Change nature examination method.
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 collagen protein sponge, gelfoam, regenerated fiber hemostatic gauze etc. it is clinical
Using many years, but different materials has the advantages that different hemostatic mechanism and corresponding drawn game limit, especially pure Animal by-product
Risk with higher.Now most sponges are gelfoam on the market, and gelatin extracts from leather more, and triple helix structure is broken
Bad, relative molecular mass distribution is wider, and it is uncontrollable not have bioactivity, material source, has been easy heavy-metal residual, and has led
Cause 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 collagen protein sponges promotes wound healing in preparation
Application in drug.
On the other hand, the object of the present invention is to provide a kind of physicochemical property detection methods of collagen protein sponge.This method
Comprehensively, effectively, gained qualified products meet national legislation standard, have the effect of excellent promotion wound healing and good production
Product quality.
In order to achieve the above object, the present invention uses following technical scheme：
The present invention provides collagen protein sponge and promotes the application in wound healing drug in preparation.
Further, the application is realized by following any one or more mechanisms of action：
A. it plays a supportive role, to form cell support arm, fetters cell, influence its migration, alienation and biosynthesis；
B. connective tissue reparation is promoted by fibroblastic aggregation；There is trend activity to surface of a wound cell, induces into fibre
It is mobile to wound site to tie up cell, vascular endothelial cell, inflammatory cell etc., the phagocytic function of activating macrophage improves body
Immunocompetence, reduce the infected chance of the surface of a wound；
C. fibre structure made of being arranged by monomer tropocollagen molecule, adsorbs blood platelet, induced platelet aggregation, and generation is released
It puts back and answers, have activated physiological hemostasis effect；
D. haemocyte is adhered to by the fibre structure of collagen, forms thrombus grumeleuse and form scab, in damaged surface of a wound blood
Barrier has been accumulated on pipe, prevents blood flow from washing damaged wound open；Enzymatic treatment can be such that antigenicity more reduces, and micro- life is isolated
Object；
F. promote impaired platelet cell release sub-cellular particles and secretion, generate thromboxane (TXA2) and blood coagulation
Enzyme；
G. growth factor is activated and be coupled, the generation and arrangement of fibroblasts and collagenous fibres are promoted, promotes meat
The generation of bud tissue；
H. activated clotting factor XII is allowed to be converted into Hageman factor a, is converted to plasma thromboplastin antecedent together with XIIa
Factor XI, plasma thromboplastin antecedent a increases the activation of the factor Ⅴ together with blood platelet, the factor Ⅴ activated in blood platelet is exposed, finally
Fibrin ferment is generated, fibrin ferment is changed into fibrin in catalysis fibre proteinogen, completes blood clotting；
I. it is finally degraded to amino acid necessary to body repair cell, provides nutrition for wound repair.
Further, the preparation method of the 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.
Preferably, in step (1), the pre-treatment is：By in the heel string through examining qualified, healthy ox fascia, fat,
Muscle picks, and is rinsed well with tap water, is cut into the thin slice that thickness is no more than 1mm；
Preferably, it in step (2), is extracted as before described：Collagen raw material after pre-treatment is put into 7-12 DEG C of purifying
It is impregnated in water, impregnates no less than 16 hours, rinsed to solution with purified water in clear state；
Preferably, in step (3), the enzymolysis liquid allocation ratio of the enzymatic hydrolysis is：750-800L purified water adds 28-32L ice
Acetic acid controls pH value in 2.5-3.5 after adding 50-55g pepsin to be sufficiently stirred；The enzymolysis step is：By what is extracted before menstruation
In collagen raw material and enzymolysis liquid investment enzymatic vessel, it is sufficiently stirred, enzymolysis time is no less than 72 hours；
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 egg is precipitated
It is white；The NaCl solution needs the filter screen filtration of 360 mesh, and strainer uses after should sterilizing, and to remove impurity, improves NaCl solution
Purity；
The dialyzate of the dialysis is phosphate buffer, and dialyzate pH value is within the scope of 2.5-3.5；The dialysis step
For：By bag filter cut into 350mm long and with purified water foam washing it is clean, then with boiling water boiling 2 hours, by the collagen for purifying of saltouing
Albumen is fitted into the bag filter for the molecular weight 12000-14000 that shuts off and dialyses；
The vacuum homogeneous is：Collagen after dialysis within the scope of pH value 5.0-5.5 is put into vacuum viscolizer, is turned
At 70-80 revs/min, vacuum degree is set in -0.9Pa, vacuum to bubble-free for number control；
Preferably, in step (4), it is described prepare collagen protein sponge the specific steps are：By the collagen after homogeneous
It is 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 physicochemical property detection method of collagen protein sponge, includes the following steps：
(1) the physical property detection of collagen protein sponge, appearance and surface texture observation including collagen protein sponge, inhales
Outlet capacity, the detection of mass per volume；
(2) the chemical property detection of collagen protein sponge, including pH value measurement, total protein, proline and hydroxyproline contain
It is fixed to measure, the measurement of residue on ignition, arsenic content, content of beary metal, pH value, readily oxidizable substance.
Further, in step (1), the appearance of the collagen protein sponge under natural light by visually observing；It is described
Surface texture is observed by scanning electron microscope (SEM)；
The detection concrete operations of the water absorbing capacity are：Collagen protein sponge is taken, electronic balance precision weighs every tablet quality,
It immerses in room temperature water, after suctioning moisture, one jiao is gently clamped with tweezers, is left the water after draining, electronic balance weighing obtains
Quality after every water suction；Calculate water absorbing capacity：Quality/sample quality after water absorbing capacity (again)=water suction；
The mass per volume is detected as：Take collagen protein sponge, length, width and the height of accurate measurement every
Degree, calculates sample volume；Electronic balance weighs quality, calculates the quality in every sponge unit volume, takes mean value.
Further, in step (1), the physical property detection further includes the detection of granularity, viscosity, and water absorbent rate contains
The calculating of water rate；
The water absorbent rate is calculated as：Sample 1g is taken, is added in the beaker for filling water, after solution saturation, is placed in
Centrifuge centrifugation, measurement sponge sucks in water centrifugation after quality, calculate water absorbent rate, refer to the absorbed moisture energy of the sponge of unit mass
Power：Q=M2/M1, Q-water absorbent rate in formula, the quality (g) after M1-sponge quality (g), M2-sponge sucks in water centrifugation；
The moisture content is calculated as：Take sample portion to weigh weight, in vacuum oven 105 DEG C to being completely dried, then
The secondary weight weighed after its drying, calculates moisture content.
Further, in step (2), the total protein content measurement is using the double of Shanghai Rongsheng Bioisystech Co., Ltd
Contracting urea method total protein content assay kit, standard protein use the collagen of Sigma company, use spirit of vinegar after accurately weighing
Dissolution；Collagen protein sponge sample is dissolved with spirit of vinegar, and lysate is taken to measure；Calculation method：Total protein content=total protein is surveyed
Definite value/collagen protein sponge quality × 100%；
The hydroxyproline content measurement uses chloramine-t method, and the hydroxyproline of Bioengineering Research Institute is built up using Nanjing
Assay kit, 6N HCl tube sealing is added after collagen protein sponge sample precise, and 110 DEG C of hydrolysis take hydrolyzate to measure；Meter
Calculation method：Hydroxyproline content=hydroxyproline determination value/total protein measured value × 100%.
Further, the appearance and surface texture are viewed as annual periodically carry out once；The measurement of the water absorbing capacity
It is periodically carried out for every half a year primary；The mass per volume is periodically to carry out once every half a year；The chemical property is detected as
It is annual periodically to carry out once.
Further, the physicochemical property detection should meet following index：
In the granularity Detection, 95% or more particle size is within the scope of 60-200 mesh；It is water-soluble in the viscosity detection
Liquid concentration is sample viscosity > 34m Pa.s under the conditions of 6.67% and 37 DEG C；The water absorbent rate reaches >=8；The moisture content
Reach≤12%；The residue on ignition reaches≤2%；The arsenic content reaches≤1mg/g；The content of beary metal reaches≤10
μg/g；In the pH value detection, the pH value of the leaching liquor of product is 5 ± 0.5；In the detection of the readily oxidizable substance, collagen egg
Bai Haimian is compared with blank, every 15g sample consumption 0.02mol/L, volume≤1.0mL of liquor potassic permanganate.
Compared with prior art, the invention has the advantages that：
(1) comprehensively, effectively, gained qualified products meet constitutional law to the physicochemical property detection method of collagen of the invention
Rule standard has the effect of excellent promotion wound healing and good product quality；
(2) collagen protein sponge of the invention can promote cell growth, differentiation, can promote connective tissue reparation, have
There is effectively hemoglutination, vascularization can be promoted, accelerates granulation tissue growth, provide nutrition abundant for the growth of tissue
Substance, accelerates the healing of injury tissue at the time for the surface of a wound that can heal in advance.
(3) appearance of collagen protein sponge product of the present invention is brought completely new general for ancient wound repair and healing
It reads, is surgeon in terms for the treatment of wound, especially cureless wound has been started a kind of safe ready and effectively treated
Method has wide clinical landscapes.
Detailed description of the invention
Fig. 1 is collagen protein sponge production technological process；
(a) and (b) of Fig. 2 is respectively the scanning electron microscope sectional view and plan view of collagen protein sponge.
Specific embodiment
Now in conjunction with attached drawing, the invention will be further described with specific embodiment.
Collagen itself is a kind of natural low immunogenicity substance, has good cell compatibility and tissue compatible
Property, angiogenesis can be promoted, degradation cycle has controllability.It can promote the differentiation of fibroblast, vascular endothelial cell
And matrix is formed, the phagocytic function of activating macrophage improves the immunocompetence of body, reduces the infected chance of the surface of a wound.?
It is widely used in the wound healings such as surface of a wound hemostasis and wound, burn and reparation.
Collagen is applied to wound trauma care by the present invention, is prepared into collagen protein sponge, can be promoted vascularization,
Accelerate granulation tissue growth, provide nutriment abundant for the growth of tissue, the time for the surface of a wound that can heal in advance accelerates
The healing of injury tissue.
Collagen protein sponge has the characteristics that for Medical wounded surface dressing：
Physics aspect：Tensile strength is high, and ductility is low, easily dry and cracked, has the morphosis of similar corium, permeable breathable
It is good；
Chemical aspect：It can carry out appropriately crosslinked, molten (solution) swollen property is adjusted, can be absorbed by tissue, it can be with drug phase interaction
With；
In terms of biology：Resist original shape, crosslinked absorption.
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.
The present invention demonstrates the biological function of collagen protein sponge and the mechanism of action to wound healing and reparation：
1, the relationship of collagen and extracellular matrix
Collagen is the main component of extracellular matrix, accounts for the one third of human body total protein, be biomass content most
High protein has extensive bioactivity, participates in cell Proliferation, movement, differentiation, it functions primarily as tissue
Support assigns tissue with tension, can be used as the packing material of shaping and beauty, it can also be used to and the pre- of artificial blood vessel coagulates,
In the diagnosis and research of many diseases, the extraction and analysis of collagen have become necessary means.
The most important function of collagen protein sponge is to play a supportive role, to form cell support arm, tissue where maintaining
Morphological integrity.Cell is followed by fettered, the functions such as its migration, alienation and biosynthesis are influenced.
The present invention is experimentally confirmed, and collagen protein sponge is by participating in the differentiation of cell and playing an important role, collagen
Protein sponge also has certain influence to growth and conversion, and collage synthesis reaction must be relied in Fibroblast cell-culture to maintain
Normal growth.The form and adhesiveness of transformed cells change all related with the reduction of collagen.
2, collagen protein sponge and connective tissue reparation
The repair ability of various tissues is different, this and the property of damage location and cell etc. are related, and usual skin injury is repaired
It is multiple very fast, this is because the fibroblastic Reproductive activity newly broken up there are skin lesion sites is strong.
Collagen protein sponge can be migration, the proliferation place mat branch of surface of a wound epidermal cell because it is with netted porous structure
Frame is conducive to the hyperplasia and reparation of cell, accelerates the healing of the surface of a wound.
3, the hemoglutination of collagen protein sponge
Collagen protein sponge is the fibre structure as made of the monomer tropocollagen molecule arrangement that molecular weight is 300kDa or so,
Fibre structure can effectively adsorb blood platelet, and then by induced platelet aggregation, generate release reaction, have activated physiological hemostasis
Effect.The grumeleuse of formation adheres on the surface of a wound of oozing of blood, plays wad act to the blood vessel of damage.Secondly, the fibre structure of collagen
Be conducive to haemocyte adherency, be formed thereon thrombus grumeleuse and form scab, accumulated barrier on damaged surface of a wound blood vessel,
Effectivelying prevent blood flow to wash open, damaged wound reaches or enzymatic treatment can be such that antigenicity more reduces, and microorganism can be isolated, biology
Compatibility is good.
In addition, collagen protein sponge can also activate and be coupled various growth factors, promote fibroblasts and glue
Generation and arrangement of fibrinogen etc., to promote the generation of granulation tissue.
Collagen and blood platelet interact in coagulation process, and blood platelet usually flows in blood, not agglutination mutually,
It is not adhered to vascular endothelial cell.In angiolysis, the eucaryotic cell structure of blood platelet changes, and is just liable to stick to vascular wall
Or extravascular substance, such as collagenous fibres, basement membrane or elastomer.Wherein, mainly collagen protein sponge can promote to be damaged
Platelet cell discharges some sub-cellular particles and secretion, including various coagulation factors, and series reaction then occurs, and generates
Thromboxane (TXA2) and fibrin ferment, TXA2 and fibrin ferment etc. be all cause blood platelet occur secretion and the stimulation of aggreation because
Son finally results in aggregation and the thrombosis of blood platelet.
The collagen-type for causing platelet aggregation, in arterial blood tube wall in addition to II Collagen Type VI, I, III, IV, collagen type v
Exist, can speculate that natural I, III, IV, collagen type v can react with blood platelet, cause the cohesion of blood platelet.
Cause the size of the collagenous fibres of platelet aggregation, not only type i collagen molecule can promote blood platelet to assemble,
AI chain also has platelet aggregation effect.The combination of blood platelet and collagen is multiple sites and original surface on platelet cell film
Upper multiple sites combine simultaneously, and this combination is combined with each other the knot for having certain degree of hardness at surface between only requiring tropocollagen molecule
Structure is strictly arranged in structure not necessarily between tropocollagen molecule without requiring in an orderly manner each other.
Also important role, collagen remove Platelet to collagen protein sponge in other links of coagulation process, promote
So that it is discharged some coagulation factors, causes the agglutination of blood platelet.Collagen can also across-the-line starting blood clotting inherent approach, including
Direct activation Hageman factor is allowed to be converted into Hageman factor a, and collagen can be such that plasma thromboplastin antecedent converts together with XIIa again
At factor XIa, furthermore can also specifically increase the activation of the factor Ⅴ together with blood platelet, make to have activated in blood platelet because
Sub- V is exposed, and finally generates fibrin ferment, and fibrin ferment is changed into fibrin in catalysis fibre proteinogen, completes blood clotting
Process.
4, the degradation of collagen protein sponge
Collagen protein sponge is as natural biomaterial, itself is non-toxic, and catabolite can be absorbed by organisms, not right
Body generates harm.Collagen protein sponge biological dressing is finally degraded to amino acid necessary to body repair cell in the surface of a wound,
Nutrition is provided for wound repair.
The preparation and characterization of 1 collagen protein sponge of embodiment
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
It can be used with alcohol wipe slicer disinfection rear.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..
The characterization of 2 collagen protein sponge of embodiment
The stability of the physicochemical property of product is to determine the important symbol of keeping life.This product is referring to external similar production
Product and the provisions of the relevant regulations issued by the State will fix tentatively 2 years validity period, while the real-time tracing carried out 4 years by a definite date to same batch products is ground
Study carefully, 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.Testing result is as shown in table 1 below.
Granularity：
By Sizing test sieve inspection technique detection：
The medicine sieve and receive container that diameter is the 200mm number of regulation are taken, weighed weight claims according to the volume density of test sample
25~100g of test sample is taken, sets in top layer's (aperture is maximum) medicine sieve and (is furnished with closely sealed reception container under undermost sieve),
It is covered on sieve.Mode of vibration and vibration frequency are set, is vibrated 5 minutes.It takes each medicine sieve and receives container, weighed weight, according to sieve
Weight differential before and after point calculates each medicine and sieves above and receive container endoparticle and powder proportion (%).It is straight to repeat aforesaid operations
To after sieving twice in succession, particle is left on each medicine sieve and the difference of powder weight leaves particle and powder weight no more than previous
5% or twice the difference of weight be not more than 0.1g；If leaving particle on a certain medicine sieve and the weight of powder taking less than test sample
The 5% of sample amount, then the weight differential of medicine sieve twice in succession should be no more than 20%.
As a result：95% or more particle size is within the scope of 60-200 mesh.
Viscosity：
It is detected by viscosmeter, as a result meets regulation：
Concentration of aqueous solution is sample viscosity > 34m Pa.s under the conditions of 6.67% and 37 DEG C.
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
Testing result is as shown in table 1 below.
Water absorbent rate：
Sample 1g is taken, is added in the beaker for filling 20mL water, after solution saturation, centrifuge centrifugation is placed in, removes more
Remaining moisture, measurement sponge sucks in water centrifugation after quality, calculate water absorbent rate.
Refer to the absorbed moisture ability of sponge of unit mass：Q-water absorbent rate in Q=M2/M1 formula, M1-sponge quality
(g), M2-sponge sucks in water centrifugation after quality (g).Its unit is g/g.
Water absorbent rate reaches >=8.
Moisture content
Sample portion is taken to weigh weight, 105 DEG C of weights to being completely dried, after weighing its drying again in vacuum oven
Amount calculates moisture content, as a result meets regulation：
Moisture content reaches≤12%.
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.
Testing result is as shown in table 1 below.
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, measure pH value of solution.
Testing result is as shown in table 2 below.
3.1.2.2. total protein 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%
Testing result is as shown in table 2 below.
3.1.2.3. the measurement of residue on ignition, arsenic content, content of beary metal, pH value, readily oxidizable substance
Residue on ignition：
Empty crucible constant weight：It takes clean crucible to set in chamber type electric resistance furnace, crucible cover is tiltedly placed on crucible, is heated to 550
DEG C blazing about 30~60 minutes, stop heating, is cooled to about 300 DEG C to temperature, takes out crucible, set in suitable drier, cover
Good crucible cover, letting cool to room temperature (generally about needs 60 minutes), and accurately weighed crucible weight is (accurately to 0.1mg).Again with same batten
Part repetitive operation, until constant weight, spare.
Weighing test sample：Test sample 5.0g is taken, is shredded.It sets in the crucible of ignition to constant weight, it is accurately weighed, and record.
Charing：The crucible for filling test sample is set on electric furnace slowly calcination (test sample burning should be avoided and overflow).It is blazing
To test sample, all charing is in black, and is no longer smoldered, and is let cool to room temperature.
Ashing：Continuation is heated to white cigarette on electric furnace and completely disappears.Crucible is set in chamber type electric resistance furnace, crucible cover is tiltedly placed on
It is about 60 minutes blazing at 550 DEG C on crucible, it is ashed test sample completely.
Constant weight：Stop heating, is cooled to about 300 DEG C to temperature, takes out crucible, set in suitable drier, cover crucible
Lid, letting cool to room temperature (generally about needs 60 minutes), and accurately weighed crucible weight is (accurately to 0.1mg).It is repeated again with similarity condition
Operation, until constant weight, and record.
It calculates：Residue on ignition %=[residue and crucible weight-empty crucible weight]/test sample weight × 100%
As a result：Residue on ignition reaches≤2%
Arsenic content：
Using microcoulomb arsenic apparatus, detection process is weighs 2 parts of potassium iodide, 0.5~1.5 part of thiocarbamide is placed in arsenic hydride hair
In raw bottle, 100~300 parts of water sample are pipetted in this bottle, and 15~30 parts of concentrated hydrochloric acid of concentration 36~37% are added, boil 3~10
It is cooling after minute, while doing reagent blank with deionized water, reagent blank is measured with microcoulomb arsenic apparatus calibrate and will be empty
White value inputs instrument, connect generating bottle with microcoulomb arsenic apparatus after water sample is cooling, hydroboration is added in arsenic hydride generating bottle
Potassium solution, arsenic apparatus carry out reaction titration automatically, measure the arsenic content in water sample.
As a result：Arsenic content reaches≤1mg/g.
Content of beary metal：
Complex reaction can occur for heavy metal and color developing agent, generate coloured molecule group, and the solution colour depth is directly proportional to concentration.
At a particular wavelength, colorimetric detection.
As a result：Content of beary metal reaches≤10 μ g/g.
PH value：
Sample 1g, 2 purified water 15mL are taken, solution ph is measured, as a result meets regulation：
The pH value of the leaching liquor of product is 5 ± 0.5.
Readily oxidizable substance：
Sample 1g is taken, water 15mL is added, adds dilute sulfuric acid 1.5mL, increases potassium manganate solution (0.02mol/L) after boiling, then boil
10min is boiled, is titrated to colourless and maintains 30s.
Every 15g sample consumption 0.02mol/L, volume≤1.0mL of liquor potassic permanganate compared with blank.
Testing result：
The testing result of collagen protein sponge physical property is as shown in table 1 below：
The testing result of 1 collagen protein sponge physical property of table
The testing result of collagen protein sponge chemical property is as shown in table 2 below：
The testing result of 2 collagen protein sponge chemical property of table
| Detection time | Testing index | Testing result |
| 2014.12 | PH value measurement | 5.35±0.31 |
| 2014.12 | Total protein content (%) | 87.3±4.22 |
| 2014.12 | Hydroxyproline content (%) | 10.78±0.56 |
| 2015.12 | PH value measurement | 5.31±0.21 |
| 2015.12 | Total protein content (%) | 86.5±4.32 |
| 2015.12 | Hydroxyproline content (%) | 10.62±0.54 |
| 2016.12 | PH value measurement | 5.00±0.49 |
| 2016.12 | Total protein content (%) | 87.5±4.32 |
| 2016.12 | Hydroxyproline content (%) | 10.54±0.50 |
| 2017.12 | PH value measurement | 5.15±0.30 |
| 2017.12 | Total protein content (%) | 88.5±4.01 |
| 2017.12 | Hydroxyproline content (%) | 10.16±0.56 |
The main part of tropocollagen molecule is rod-shaped triple-helix structure.
Experiment shows that restricted pepsin acetic acid extracts collagen, and by experimental analysis, tissue is dropped through pepsin
Xie Hou, in giant molecule structure, the form of a large amount of collagen spun gold entire molecules is released, and is dissolved in acetic acid.Collagen
Pepsin selective hydrolysis can be endured, collagen is not susceptible to be denaturalized under cryogenic, and triple-helix structure has been kept
It is good.
The physicochemical property and biological property of collagen protein sponge：
(1) physicochemical property：
A. product appearance is creamy white sheet, and one-sided smooth is coarse on one side, easy to clean, inviolateness；
B. the water absorbing force of product is not less than 10 times of own wt；
C. the content of beary metal of product is not more than 2 μ g/g；
D. the pH value of product is not less than 4.0；
(2) biological property：
A. product no cytotoxicity；
B. product is without hypersensitive；
C. product is without skin irritation；
D. product is sterile.
Effete test embodiment
Treatment evaluation to rat back full thickness dermal wounds：
Choosing weight is healthy rat 60 of 180~220g, and 3% yellow Jackets (30mg/kg) is injected intraperitoneally at it,
It is divided into 3 groups, every group 20 at random.The surface of a wound of two 2.0cm*2.0cm is respectively cut in rat back backbone on both sides of the middle, and is broken
Bad a little musculature, it is spare after hemostasis.
Sample is pasted at the surface of a wound, next day dressing.It photographs to record within the 2nd day, the 5th day and the 10th day after the surface of a wound is made
Wound healing situation measures surface of a wound area with analysis image software I magepro-Plus, and statistics obtains rat back full thickness skin
Wound Defect healing rate, is shown in Table 3：
3 rat back Repair of Cutaneous Full-thickness Excision rate of table
| Group | 2nd day | 4th day | 7th day |
| Embodiment 1 | 29.86% | 85.23% | 98.76% |
| Commercially available medical sponge | 14.05% | 48.07% | 87.85% |
It can be seen that from the result of table 3, the collagen protein sponge of embodiment 1 uniformly preferably promotes to heal to the surface of a wound of rat
Effect, effect are significantly better than commercially available medical sponge, illustrate that collagen protein sponge of the invention can promote tissue growth and the surface of a wound is cured
It closes.
Detect influence of the leaching liquor of collagen protein sponge to 2,4,7 days opposite proliferation rates of fibroblast：
Being made into concentration with the Mouse Skin Fibroblasts (2-3 generation) of cell culture fluid logarithmic growth phase is 6 × 104A/
ML cell suspension, every 100 μ L of hole, is inoculated in 96 well culture plates, sets 37 DEG C, 5%CO2It is cultivated 24 hours in incubator；Respectively
(200 hole μ L/) is replaced with the material leaching liquor of 50%, 100% concentration, using cell culture fluid as blank control, continues to set 37 DEG C
Culture overturns away liquid in 2 holes after 4 hours, every hole adds 150 μ L of DMSO, shakes 10min；Enzyme linked immunological instrument measures every hole
Absorbance value, each group 8 take mean value.Measurement wavelength is 490nm, presses formula R=experimental group OD/ control group OD × 100% later
Calculate the cell opposite proliferation rate (RGR) of sample.
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.
Implantation Test：
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.
By above-mentioned analysis, collagen protein sponge has remarkable effect to wound repair and healing, has to surface of a wound cell
To activity, induced fibroblast, vascular endothelial cell, inflammatory cell etc. are mobile to wound site, and activating macrophage gulps down
Function is bitten, the immunocompetence of body is improved, reduces the infected chance of the surface of a wound.
Collagen is the somatomedin of fibroblast, vascular endothelial cell etc., can promote vascularization, accelerates meat
Bud tissue growth provides nutriment abundant for the growth of tissue, and fibroblast is also the main cell for synthesizing collagen,
And collagen is the main component of cytoplasm in granulation tissue.Therefore, collagen protein sponge can heal the time of the surface of a wound in advance,
Accelerate the healing of injury tissue.
To sum up, collagen protein sponge has the function of hemostasis, promotes wound healing.Collagen protein sponge is for creating
Blood platelet is adsorbed in the early stage (inflammation phase) in face, interacts with coagulation factor, and cause platelet aggregation, plays hemostasis and makees
With；The destruction of blood platelet makes it release various active substance, such as growth factor, starts wound healing；Induce fibroblast
Activity, the function of different haemocytes is activated and adjusts, including phagocytosis and taxis.In intergrade (granulation tissue
Growth period), promote regeneration, the arrangement of internal collagen, enhances the good haemostatic effect of exudate；When collagen protein sponge and bleeding
When the surface of a wound contacts, blood can be adsorbed rapidly using the capillarity of its large area sponge structure, cause platelet aggregation, and then broken
Bad blood platelet, activation and release coagulation factor, to promote coagulation process；Simultaneously sponge the surface of a wound dissolution or degradation and generate
Local viscosity variation, can also promote coagulation process.In later period (mature recovery period), the bracket of epithelial cell is formed, is induced into fibre
The generation and arrangement for tieing up cell and different collagenous fibres cell, promote the generation of granulation tissue；Promote blood vessel and newborn cicatricial tissue
Formation, reach wound healing.It is absorbed finally, collagen protein sponge material is degraded by body.
From clinical data as can be seen that in surgical wound surface and residual cavity filling hemostasis, collagen protein sponge and the bleeding surface of a wound
One contact, contact surface quickly form gel and are adhered on the bleeding surface of a wound, and outside then forms continuous thousand layers of compressing, establish one
The ideal haemostatic state of kind and structure.
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. collagen protein sponge promotes the application in wound healing drug in preparation.
2. application according to claim 1, which is characterized in that the application passes through following any one or more mechanisms of action
It realizes：
A. it plays a supportive role, to form cell support arm, fetters cell, influence its migration, alienation and biosynthesis；
B. connective tissue reparation is promoted by fibroblastic aggregation；There is trend activity to surface of a wound cell, induces into fiber finer
Born of the same parents, vascular endothelial cell, inflammatory cell etc. are mobile to wound site, and the phagocytic function of activating macrophage improves exempting from for body
Epidemic disease activity, reduces the infected chance of the surface of a wound；
C. fibre structure made of being arranged by monomer tropocollagen molecule, adsorbs blood platelet, and it is anti-to generate release for induced platelet aggregation
It answers, has activated physiological hemostasis effect；
D. haemocyte is adhered to by the fibre structure of collagen, forms thrombus grumeleuse and form scab, on damaged surface of a wound blood vessel
Barrier has been accumulated, has prevented blood flow from washing damaged wound open；Enzymatic treatment can be such that antigenicity more reduces, and microorganism is isolated；
F. promote impaired platelet cell release sub-cellular particles and secretion, generate thromboxane and fibrin ferment；
G. growth factor is activated and be coupled, the generation and arrangement of fibroblasts and collagenous fibres are promoted, promotes granulation group
The generation knitted；
H. activated clotting factor XII is allowed to be converted into Hageman factor a, so that plasma thromboplastin antecedent is converted to the factor together with XIIa
XIa increases the activation of the factor Ⅴ together with blood platelet, the factor Ⅴ activated in blood platelet is exposed, final to generate
Fibrin ferment, fibrin ferment are changed into fibrin in catalysis fibre proteinogen, complete blood clotting；
I. it is finally degraded to amino acid necessary to body repair cell, provides nutrition for wound repair.
3. application according to claim 1, which is characterized in that the collagen protein sponge is through the following steps that be prepared into
It arrives：
(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.
4. application according to claim 3, it is characterised in that：
In step (1), the pre-treatment is：Fascia, fat, muscle in heel string through examining qualified, healthy ox is picked, is used
Tap water is rinsed well, and the thin slice that thickness is no more than 1mm is cut into；
In step (2), it is extracted as before described：Collagen raw material after pre-treatment is put into 7-12 DEG C of purified water and is impregnated, is soaked
It bubble no less than 16 hours, is rinsed to solution with purified water in clear state；
In step (3), the enzymolysis liquid allocation ratio of the enzymatic hydrolysis is：750-800L purified water adds 28-32L glacial acetic acid, adds 50-
Control pH value is in 2.5-3.5 after 55g pepsin is sufficiently stirred；The enzymolysis step is：The procollagen that will be extracted before menstruation
In material and enzymolysis liquid investment enzymatic vessel, it is sufficiently stirred, enzymolysis time is no less than 72 hours；
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 dialyzate of the dialysis is phosphate buffer, and dialyzate pH value is within the scope of 2.5-3.5；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 hours, by the collagen egg for purifying of saltouing
It dialyses in the white bag filter for being fitted into the molecular weight 12000-14000 that shuts off；
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；
In step (4), it is described prepare collagen protein sponge the specific steps are：Collagen after homogeneous is packed into sterilized
In lyophilized plate, then lyophilized plate is fitted into drying box of freeze dryer；Product is chilled to -40 DEG C hereinafter, heat preservation no less than 4 from room temperature
Hour, into vacuum operation, it is heated to 30-40 DEG C after being warming up to 5 DEG C naturally under vacuum state, makes product at platinum sponge
Sheet.
5. a kind of physicochemical property detection method of collagen protein sponge, which is characterized in that include the following steps：
(1) the physical property detection of collagen protein sponge, appearance and surface texture observation including collagen protein sponge, water absorbing capacity
Power, the detection of mass per volume；
(2) the chemical property detection of collagen protein sponge, including pH value measurement, total protein and hydroxyproline content measure, blazing
The measurement of residue, arsenic content, content of beary metal, pH value, readily oxidizable substance.
6. physicochemical property detection method according to claim 5, it is characterised in that：
In step (1), the appearance of the collagen protein sponge under natural light by visually observing；The surface texture is by sweeping
Retouch Electronic Speculum observation；
The detection concrete operations of the water absorbing capacity are：Collagen protein sponge is taken, electronic balance precision weighs every tablet quality, immerses
In room temperature water, after suctioning moisture, one jiao gently being clamped with tweezers, is left the water after draining, electronic balance weighing obtains every
Quality after water suction；Calculate water absorbing capacity：Quality/sample quality after water absorbing capacity=water suction；
The mass per volume is detected as：Take collagen protein sponge, length, width and the height of accurate measurement every, meter
Calculate sample volume；Electronic balance weighs quality, calculates the quality in every sponge unit volume, takes mean value.
7. physicochemical property detection method according to claim 5, it is characterised in that：
In step (1), the physical property detection further includes the detection of granularity, viscosity, the calculating of water absorbent rate, moisture content；
The water absorbent rate is calculated as：Sample 1g is taken, is added in the beaker for filling water, after solution saturation, is placed in centrifugation
Machine centrifugation, measurement sponge sucks in water centrifugation after quality, calculate water absorbent rate, refer to the absorbed moisture ability of the sponge of unit mass：Q
=M2/M1, Q-water absorbent rate in formula, the quality after M1-sponge quality, M2-sponge sucks in water centrifugation；
The moisture content is calculated as：Take sample portion to weigh weight, in vacuum oven 105 DEG C to being completely dried, claim again
Weight after measuring its drying, calculates moisture content.
8. physicochemical property detection method according to claim 5, it is characterised in that：
In step (2), the total protein content measurement uses the biuret method total protein of Shanghai Rongsheng Bioisystech Co., Ltd
Assay kit, standard protein use the collagen of Sigma company, are dissolved after accurately weighing with spirit of vinegar；Collagen egg
White sponge sample is dissolved with spirit of vinegar, and lysate is taken to measure；Calculation method：Total protein content=total protein measured value/collagen egg
Bai Haimian mass × 100%；
The hydroxyproline content measurement uses chloramine-t method, and 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, and 110 DEG C of hydrolysis take hydrolyzate to measure；Calculating side
Method：Hydroxyproline content=hydroxyproline determination value/total protein measured value × 100%.
9. physicochemical property detection method according to claim 5, it is characterised in that：
The appearance and surface texture are viewed as annual periodically carry out once；
Being measured as the water absorbing capacity periodically carries out once every half a year；
The mass per volume is periodically to carry out once every half a year；
The chemical property is detected as annual periodically carry out once.
10. physicochemical property detection method according to claim 7, which is characterized in that the physicochemical property detection should accord with
Close following index：
In the granularity Detection, 95% or more particle size is within the scope of 60-200 mesh；
In the viscosity detection, concentration of aqueous solution is sample viscosity > 34m Pa.s under the conditions of 6.67% and 37 DEG C；
The water absorbent rate reaches >=8；
The moisture content reaches≤12%；
The residue on ignition reaches≤2%；
The arsenic content reaches≤1mg/g；
The content of beary metal reaches≤10 μ g/g；
In the pH value detection, the pH value of the leaching liquor of product is 5 ± 0.5；
In the detection of the readily oxidizable substance, collagen protein sponge is compared with blank, every 15g sample consumption 0.02mol/L, permanganic acid
Volume≤1.0mL of potassium solution.
Priority Applications (1)
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| CN111595953A (en) * | 2020-05-22 | 2020-08-28 | 盐城工学院 | Dialysis method based calculation method for substance adsorption amount and desorption amount of extracellular polymer |
| CN114632183A (en) * | 2022-03-30 | 2022-06-17 | 温州医科大学 | Wound protein sponge, preparation method thereof and application of wound protein sponge in preparation of medicines for reducing scar formation in skin repair |
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