CN104667336B

CN104667336B - Human body biological dressing for covering burn wound surfaces and applications of dressing

Info

Publication number: CN104667336B
Application number: CN201510094897.3A
Authority: CN
Inventors: 孙继煌; 王春元; 石清东; 吴文华
Original assignee: BEIJING JAYYALIFE BIOLOGICAL TECHNOLOGY Co Ltd
Current assignee: BEIJING JAYYALIFE BIOLOGICAL TECHNOLOGY Co Ltd
Priority date: 2013-03-28
Filing date: 2013-03-28
Publication date: 2017-01-25
Anticipated expiration: 2033-03-28
Also published as: CN104667336A

Abstract

The invention provides a human body biological dressing for covering burn wound surfaces and applications of the dressing. Antigen removal and immunogenicity reduction treatment is performed on allograft with biological means, biochemical means and the like, dermis and epidermis are kept, and the clinical effect is far better than that of the allograft. A preparation method of the dressing sequentially comprises steps as follows: soaking treatment with a glutaraldehyde solution; segmentation; soaking treatment with purified water; soaking treatment with a polysorbate 80 solution; soaking treatment with purified water; soaking treatment with an amino acid solution; soaking treatment with purified water; soaking treatment with PBS (phosphate buffered saline); repair and trimming; soaking treatment with a sodium chloride solution; packaging and irradiation. The obtained dressing can purify and clean the wound surfaces, promote growth of granulation tissue, cover granulation wound surfaces, chronic ulcers and bedsores, shorten healing time and be applied to the field of rescue of large-area deep burns, coverage of various burn (wound) surfaces, coverage and treatment of excision surfaces, granular skin-grafting wound surfaces, the granulation surfaces, the chronic ulcers and the bedsores and the like.

Description

A kind of human-body biological dressing for covering the burn trauma surface of a wound and its application

The application is to be on March 28th, 2013 applying date, Application No. 201310103371.8, invention entitled " a kind of The divisional application of the Chinese invention patent application of the preparation method for covering the human-body biological dressing of the burn trauma surface of a wound ".

Technical field

The present invention relates to a kind of burn trauma surface of a wound covering material and its application, particularly one kind is used for covering burn trauma The allograft skin of the surface of a wound goes antigen to reduce immunogenic human-body biological dressing and its concrete application.

Background technology

Burn is at ordinary times and during the war years most common skin trauma, and the various infringements that burnt degree causes are all and skin barrier The destruction of effect is relevant with forfeiture.As lost in: metabolic aggravation, temperature decline, excessive water, a large amount of loss of protein and Endocrine and immune imbalance.Therefore use dressing flap coverage, as interim body surface protective barrier, wound healing, Especially large-area burns are particularly important.People early find that biomaterial contributes to the healing of skin trauma.With right Constantly deeply, people gradually recognize that purpose using dressing, much not only for flap coverage, is applied to researching wound healing Material also should be able to wound healing.1962, doctor winter of London University delivered famous paper on nature magazine, Demonstrate the Healing to the surface of a wound for the moist environment.Compared with polyethylene film closing pig partial thickness skin Wound Defect, it is exposed to The surface of a wound re-epithelialization probability of in the air increases by 50%, and subsequent a large amount of both at home and abroad clinical practices and basic research all support this Idea.

Nearly 4o to have developed multiple temporary surface of a wound both at home and abroad and to have covered dressing, and is widely used in clinic.At present The temporary surface of a wound of Clinical practice covers dressing and specifically includes that traditional dressing, synthetic dressing, natural biological dressing three major types.And The selection of burn wound covering material is burn treating, particularly important in a large-area burns trauma care ring.

Traditional dressing such as gauze, cotton pad etc. have the disadvantage in that and the surface of a wound 1. cannot be kept to moisten, wound healing postpones；② Dressing fiber is easy to fall off, causes foreign body reaction, impact healing；3. wound granulation is organized in the mesh of dressing of easily growing into, during dressing Pain can be caused；4., when dressing is saturated, pathogen is easily passed through；5. during dressing, the newborn tissue of easy damaged；6. dressing workload Greatly.

In synthetic dressing, the film class synthetic dressing product such as such as tegaderm, dermafilm, oprafiex, opsite Water vapour permeability too low, easy hydrops, can induce or increase infect.Foam type synthetic dressing such as allevyn, ixralo etc. Product hole is big, and wound granulation tissue is easily grown into, and causes demoulding difficult thus bringing wound again, and is easily subject to germ contamination, easily Leave detritus in the surface of a wound.The aerosol type synthetic dressing product such as such as hydron, aeroplast has the disadvantage in that adhesiveness and resists Zhang Qiangdu is poor；Moisture retention is poor, and surface of a wound water evaporation quantity is big；The water solubility of spraying film can make it be softened by wound exudate, keeps Time is short；, easily there is infection under film for polluting the surface of a wound in no infection control effect.Hydrogels dressing such as duoderm, The mechanical performance of the products such as comfeel, restore, intrasite is too poor, a large amount of absorb exudates after can be swollen because of colloid Swollen and lead to dressing and wound separation, provide chance to the intrusion breeding of bacterium.

Natural biological dressing include autologous skin, alloskin (abbreviation allograft skin), radiated pig skin (xenogenesis skin), amnion, Cell-less corium ground substance, collagen class dressing etc..The optimal method of flap coverage is autologous transplanting skin at present, but burn surface area Patients with Big Area Burn more than 50%, autologous skin source seems wretched insufficiency.Skin allograft is to be proved relatively over nearly 40 years For one of effective skin substitute products, it is a kind of more satisfactory wound-surface cover, there is preferably skin barrier function.Allosome The penetrability of skin, adhesiveness are similar to autologous skin, can stop bacterial invasion and stop surface of a wound water, electrolyte, protein and heat The loss of amount, and there is good hemostasis and promote epithelialization function.

The existing very long history of the application in burn field for the allograft skin, has mitigation patient suffering in burn treating, promotees Enter the effect of wound healing.Cochrane in 1966 earliest by stored frozen allograft skin (cryopreserved allograft, Cpa) it is applied to burn wound to succeed, but the process of cpa and preservation are fairly cumbersome, and antigenicity is stronger, rejection is fast, is facing Cannot popularization and application on bed.The glycerine of latter improvement in 1984 preserves allograft skin (glycerol preserved Allograft, gpa) start clinically to apply, ratio skin-deep with cpa, gpa is readily produced and preserves, and antigenicity is relatively low, anti-sense Dye ability is also relatively strong, and the time-to-live significantly extends, and has clinically obtained wide popularization and application.But existing allosome at present There is preservation condition and have high demands, antigenicity, have the clinical problems such as occupancy, pathogenic microorganism easy infection in skin.Especially because its The antigenicity that exists and the obvious immunological rejection that causes, general 2-3 week left and right dissolving, come off, and the surface of a wound is exposed, affects a phase Healing.Recently as the development of biotechnology, more new allograft skin human-body biological dressing starts clinical into people.

Content of the invention

In order to overcome the deficiencies in the prior art, the invention provides a kind of human-body biological for covering the burn trauma surface of a wound Dressing, the present invention, by the use of allograft skin as raw material, has carried out removing antigen to allograft skin by means such as biology, biochemistries Reduce immunogenicity to process, remain corium and epidermis, compared with gpa, its immunological rejection postpones, application and preservation are more Easy.Product has not only reached the purpose of early stage flap coverage, but also can wound healing, clinical practice does not find bright Aobvious immunological rejection and systemic inflammatory response.

The technical solution adopted for the present invention to solve the technical problems is: a kind of human body for covering the burn trauma surface of a wound Biological dressing semi-finished product and the human-body biological dressing covering the burn trauma surface of a wound, wherein, the preparation of human-body biological dressing semi-finished product Method comprises the steps 1-10, and the preparation method of human-body biological dressing comprises the steps 1-11, specially carries out in order The following step:

Step [1] is 0.25%- in the mass fraction that the ratio that allograft skin every square centimeter takes 0.5-3ml takes respective amount 3% glutaraldehyde solution, allograft skin is put in taken glutaraldehyde solution, soaks 15-60 minute；Change glutaraldehyde solution, continue Continuous immersion 15-30 minute；Take out allograft skin, divide in the quality that the ratio that allograft skin every square centimeter takes 0.5-3ml takes respective amount The sodium chloride solution for 0.5%-1.5% for the number, allosome ratio is put in taken sodium chloride solution, soaks 30-120 minute, takes out Allograft skin obtains semi-finished product a；

Semi-finished product a is split by step [2] by required specification, with taking skin drum that the semi-finished product a after segmentation is carried out anti-take, Remove the adipose tissue of remaining, obtain semi-finished product b；

Step [3] takes the ratio of 0.5-3ml to take the purified water of respective amount in semi-finished product b every square centimeter, by semi-finished product b and Taken purified water is placed in same container, is positioned over shaking table concussion 15-60 minute, changes purified water, is again placed in shaking table concussion 15-60 minute.Repeat above step, until when the ph value of the purified water displacing is between 5.5-6.5, discarding purified water, obtaining Semi-finished product c；

It is 0.05%- that step [4] takes the ratio of 0.5-3ml to take the mass fraction of respective amount in semi-finished product c every square centimeter 0.3% polyoxyethylene sorbitan monoleate solution, semi-finished product c and taken polyoxyethylene sorbitan monoleate solution is placed in same container, is positioned over shaking table Concussion 15-60 minute, changes polyoxyethylene sorbitan monoleate solution, is again placed in shaking table concussion 15-60 minute.Repeat above step, until When the ph value of the polyoxyethylene sorbitan monoleate solution displacing is between 6.0-7.0, discard polyoxyethylene sorbitan monoleate solution, obtain semi-finished product d；

Step [5] takes the ratio of 0.5-3ml to take the purified water of respective amount in semi-finished product d every square centimeter, by semi-finished product d and Taken purified water is placed in same container, is positioned over shaking table concussion 15-60 minute, changes purified water, is again placed in shaking table concussion 15-60 minute.Repeat above step, until when the ph value of the purified water displacing is between 5.5-6.5, discarding purified water, obtaining Semi-finished product e；

Step [6] takes the ratio of 0.5-3ml to take the Freamine Ⅲ of respective amount in semi-finished product e every square centimeter, will half one-tenth Product e and taken Freamine Ⅲ are placed in same container, put shaking table concussion 15-60 minute, then stand 15-30 minute, discard Freamine Ⅲ, obtains semi-finished product f；

Step [7] takes the ratio of 0.5-3ml to take the purified water of respective amount in semi-finished product f every square centimeter, by semi-finished product f and Taken purified water is placed in same container, is positioned over shaking table concussion 15-60 minute, changes purified water, is again placed in shaking table concussion 15-60 minute.Repeat above step, until when the ph value of the purified water displacing becomes between 6.0-7.0, discarding purified water, Obtain semi-finished product g；

Step [8] takes the ratio of 0.5-3ml to take the pbs buffer solution of respective amount in semi-finished product g every square centimeter, by semi-finished product G and taken pbs buffer solution are placed in same container, are positioned over shaking table concussion 15-60 minute, then stand 15-30 minute, discard The pbs buffer solution being used, obtains semi-finished product h；

Step [9] will in the corium face of semi-finished product h, epidermis side all foreign matters remove clean, if exist a diameter of 1 centimetre with On hole, then with medical suture, hole is sewed on, if shape is irregular, by its trimmed, obtains semi-finished product i；

It is 0.5%- that step [10] takes the ratio of 0.5-3ml to take the mass fraction of respective amount in semi-finished product i every square centimeter 1.5% sodium chloride solution, then semi-finished product i and taken sodium chloride solution is placed in same container, is positioned over shaking table concussion 15-60 minute, changes sodium chloride solution, is again placed in shaking table concussion 15-60 minute.Repeat above step, until displace When the ph value of sodium chloride solution is between 6.5-7.5, take out semi-finished product i；Take the ratio of 1ml by every 1-3 square centimeter semi-finished product i Example takes the sodium chloride solution that the mass fraction of respective amount is 0.5%-1.5%, semi-finished product i and taken sodium chloride solution is placed in same In one container, it is standing and soak for 15-60 minute, discards solution, obtain semi-finished product j；

Semi-finished product j is loaded packing inside bag by step [11], paves, packing inside bag is sealed, then fill in packing inside bag Enter outer packaging bag and be sealed against, after through cobalt co⁶⁰Irradiation, final finished product k.

Allograft skin described in above-mentioned steps [1] derives from the skin that healthy human body contributes body, and its thickness is 0.2-0.3 milli Rice, the thickness difference between taken allograft skin is less than 0.1 millimeter.

Freamine Ⅲ described in above-mentioned steps [6], amino acid contained therein is glutamic acid, alanine, glycine, One or several in the middle of asparatate, in the middle of every 1000 milliliters of Freamine Ⅲs, glutamic acid, alanine, glycine, Tianmen The content of one or several of winter propylhomoserin is respectively 0.1-3g respectively.

The ph value of the pbs buffer solution described in above-mentioned steps [8] is 7.0-8.0.

Met with medical suture described in above-mentioned steps [9] and the distance between stitch during hole, should be controlled to be not more than 1 centimetre.

Through cobalt co in above-mentioned steps [11]⁶⁰The dosage of irradiation is: 20kgy≤irradiation dose≤30kgy.

The positive effect of the present invention: the human-body biological dressing of gained of the present invention intactly remains the three-dimensional of extracellular matrix Morphosis and composition (growth framework), inducing host cell can grow in order, not only shorten wound healing time, also improve Wound healing quality, decreases cicatrization；Deep burn mesh graft application its immunological rejection of products obtained therefrom of the present invention Reaction is low, effectively extends the cover time, it is to avoid the postoperative exposed too early surface of a wound, it is therefore prevented that trauma surface infestation and PD, is Later by stages autologous skin-grafting creates condition, thus the application being more beneficial in extensive deep burn is given treatment to.

Product of the present invention and wounds have good compatibility, can effectively completely cut off the surface of a wound and extraneous directly contact, Serve temporary transient skin barrier effect, be that wound healing provides a good repairing environment, be conducive to epithelial cell to give birth to Long, shorten wound healing time and improve the quality after wound healing.

Using the method for the invention be obtained product be applied to all kinds of burnings (wound) hinder the surface of a wound covering it is adaptable to shallow °, Deep °, ° cut the covering treatment of (cutting) scab surface of a wound, the Micrograiting surface of a wound, granulation wound and chronic ulcer and bedsore.This product Have skin complete remove antigen skin corium and epidermal area, while reducing immunological rejection, remain skin corium with The corresponding function of epidermal area, covers to hinder in burning (wound) and can play temporary transient skin barrier effect on the surface of a wound, can be longer on wound base Surviving of time and no substantially occupy-place, especially cover extensive deep burn and cut (cutting) scab Micrograiting surface of a wound, can heal a phase Merge and improve wound repair quality.

Brief description

Fig. 1 is a kind of flow chart of the preparation method of the human-body biological dressing for covering the burn trauma surface of a wound of the present invention.

Fig. 2 virus inactivation technology of the present invention (0.5% glutaraldehyde solution immersion treatment) hiv Inactivation Dynamics curve.

Fig. 3 uses the change of different time rabbit anteserum igg level after human-body biological dressing process of the present invention.

Fig. 4 uses the change of different time rabbit anteserum iga level after human-body biological dressing process of the present invention.

Fig. 5 uses the change of different time rabbit anteserum igm level after human-body biological dressing process of the present invention.

Before Fig. 6 experiment, each group Serum Antibodies of Rabbits level compares.

After Fig. 7 is processed using human-body biological dressing of the present invention, 2 weeks each group Serum Antibodies of Rabbits levels compare.

After Fig. 8 is processed using human-body biological dressing of the present invention, 4 weeks each group Serum Antibodies of Rabbits levels compare.

After Fig. 9 is processed using human-body biological dressing of the present invention, 12 weeks each group Serum Antibodies of Rabbits levels compare.

Specific embodiment

Below in conjunction with the accompanying drawings to a preferred embodiment of the present invention will be described in detail.

As shown in figure 1, the preferred embodiment of the present invention provides a kind of system of the biological dressing for covering the burn trauma surface of a wound Make method, the following step including carrying out in order:

Step [1] glutaraldehyde solution immersion treatment: (contribute the skin of body from healthy human body by allograft skin every square centimeter Skin, its thickness is 0.2-0.3 millimeter) take the glutaraldehyde solution that the ratio of 1ml takes the mass fraction of respective amount to be 0.5%, will be different Body skin is put in taken glutaraldehyde solution, soaks 50 minutes；Change glutaraldehyde solution, continue to soak 25 minutes；Take out allograft skin, The sodium chloride solution being 0.9% in the mass fraction that the ratio that alloskin every square centimeter takes 1ml takes respective amount, by allosome Skin is put in taken sodium chloride solution, soaks 90 minutes, takes out allograft skin and obtains semi-finished product a；

Step [2] is split: semi-finished product a is split by required specification, with taking skin drum that the semi-finished product a after segmentation is carried out The anti-adipose tissue taking, removing remaining, obtains semi-finished product b；

Step [3] purified water immersion treatment: take the ratio of 2ml to take the purified water of respective amount in semi-finished product b every square centimeter, Semi-finished product b and taken purified water are placed in same container, are positioned over shaking table and shake 30 minutes, change purified water, be again placed in Shaking table shakes 30 minutes.Repeat above step, until when the ph value of the purified water displacing is between 5.5-6.5, discarding purifying Water, obtains semi-finished product c；

Step [4] polyoxyethylene sorbitan monoleate solution immersion treatment: take the ratio of 2ml to take respective amount in semi-finished product c every square centimeter Mass fraction be 0.2% polyoxyethylene sorbitan monoleate solution, semi-finished product c and taken polyoxyethylene sorbitan monoleate solution are placed in same container In, it is positioned over shaking table and shakes 30 minutes, change polyoxyethylene sorbitan monoleate solution, be again placed in shaking table and shake 30 minutes.Repeat above walking Suddenly, until when the ph value of the polyoxyethylene sorbitan monoleate solution displacing is between 6.0-7.0, discarding polyoxyethylene sorbitan monoleate solution, obtaining half one-tenth Product d；

Step [5] purified water immersion treatment: take the ratio of 2ml to take the purified water of respective amount in semi-finished product d every square centimeter, Semi-finished product d and taken purified water are placed in same container, are positioned over shaking table and shake 30 minutes, change purified water, be again placed in Shaking table shakes 30 minutes.Repeat above step, until when the ph value of the purified water displacing is between 5.5-6.5, discarding purifying Water, obtains semi-finished product e；

Step [6] Freamine Ⅲ immersion treatment: in semi-finished product e every square centimeter take the ratio of 2ml take respective amount by The amino acid mixed solution that glutamic acid and glycine mix, contains glutamic acid in every 1000 milliliters this amino acid mixed solutions 0.75g, glycine 1.5g, semi-finished product e and taken amino acid mixed solution are placed in same container, put shaking table and shake 30 points Clock, then stands 30 minutes, discards amino acid mixed solution, obtain semi-finished product f；

Step [7] purified water immersion treatment: take the ratio of 2ml to take the purified water of respective amount in semi-finished product f every square centimeter, Semi-finished product f and taken purified water are placed in same container, are positioned over shaking table and shake 30 minutes, change purified water, be again placed in Shaking table shakes 30 minutes.Repeat above step, until when the ph value of the purified water displacing is changed between 6.0-7.0, discarding Purified water, obtains semi-finished product g；

Step [8] pbs buffer solution immersion treatment: take the ratio of 2ml to take the respective amount ph value to be in semi-finished product g every square centimeter 7.5 pbs buffer solution, semi-finished product g and taken pbs buffer solution are placed in same container, are positioned over shaking table concussion 30 minutes, so Stand 30 minutes afterwards, discard used pbs buffer solution, obtain semi-finished product h；

Step [9] is repaired and finishing: foreign matters all in the corium face of semi-finished product h, epidermis side is removed clean, if existing straight Footpath is more than 1 centimetre of hole, then sewed on hole with medical suture, controls the distance between stitch to be not more than 1 during slot apertures hole Centimetre, if shape is irregular, by its trimmed, obtain semi-finished product i；

Step [10] sodium chloride solution immersion treatment: take the ratio of 1ml to take the matter of respective amount in semi-finished product i every square centimeter Amount fraction is 0.9% sodium chloride solution, then semi-finished product i and taken sodium chloride solution is placed in same container, is positioned over Shaking table shakes 30 minutes, changes sodium chloride solution, is again placed in shaking table and shakes 30 minutes.Repeat above step, until displacing Sodium chloride solution ph value between 6.5-7.5 when, take out semi-finished product i；Take the ratio of 1ml in every 1 square centimeter of semi-finished product i Take respective amount mass fraction be 0.9% sodium chloride solution, semi-finished product i and taken sodium chloride solution are placed in same container In, it is standing and soak for 30 minutes, discards solution, obtain semi-finished product j；

Step [11] packaging and radiation sterilization: semi-finished product j is loaded packing inside bag, paves in packing inside bag, by interior bag Pack sealing, is then charged into outer packaging bag and is sealed against, and finally irradiated dosage is the cobalt co of 25kgy⁶⁰Irradiation, finally must become Product k.

First, allograft skin raw material in step described in above-described embodiment [1] are inactivated through glutaraldehyde solution immersion treatment technique The checking of hiv effect is as follows:

1 material

1.1 raw material skins, i.e. allograft skin raw material described in above-described embodiment step [1]: lot number 1,2,3；

1.2 50% glutaraldehydes (are configured to 0.5% using front deionized water)；

1.3 human immunodeficiency viruses: hiv-1 iiib strain, introduced by the U.S.；

1.4 mt₂Cell: pass on people's t lymphocytic series；

1.5 cell culture fluids: rpmi 1640+10% hyclone；

1.6 96 porocyte culture plates

1.7 co₂Incubator: mco-15a.

2 methods

Take each one piece of (the about 1cm of raw material skin respectively²/ block), pbs cyclic washing, abandon pbs, add hiv-1 iiib 0.1ml, after repeatedly blowing and beating several times, is incubated 30min, adds rpmi 1640 cell culture fluid 0.9ml, mix, move to filter centrifugation Guan Zhong, 3000 revs/min 2 minutes, filtrate as when zero sample；Take each one piece of (the about 1cm of raw material skin more respectively²/ block), pbs is anti- After backwashing is washed, and abandons pbs, adds hiv-1 iiib 0.1ml, after repeatedly blowing and beating several times, is incubated 30min, adds 0.5% penta 2 Aldehyde 0.9ml, immersion treatment 50 minutes, moved in filtering centrifuge tube respectively at 10,20,30,50 minutes, 3000 revs/min 2 points Filtrate is sampled by clock respectively；Every sub-sampling 0.1ml, makes 10 gradient dilution at once of nutrient solution, measures the wherein titre of hiv, Set virus control, dilution comparison, positive control and cell controls simultaneously, measure the titre of hiv with mt2 cell micro cultivation, Using cytopathy as the index differentiating that virus exists, 1 batch sample is carried out with three duplicate detection, 2 and 3 batch samples respectively carry out one Secondary detection.

3 conclusions

Allograft skin raw material described in step of the present invention [1] add hiv-1 iiib, are incubated 30 minutes, add 0.5% glutaraldehyde immersion treatment 50 minutes, can make the titre of hiv-1 iiib decline 4.17 log (testing result such as Fig. 2 institutes Show) and through cell culture blind passage three generations, cytopathy does not occur.

2nd, as follows to production technology Validation of Virus Inactivation in Human of the present invention:

Checking foundation: traditional Chinese medicines prison notes [2002] No. 160, " animal derived medical device product registration declaration material writes finger Lead principle ", " allogeneic medicine equipment virus inactivation technology verification guide principle ".

Verifying purpose: according to sample manufacturing process, separately verify 0.5% glutaraldehyde immersion 50 minutes, Co 60-γ penetrates The inactivating efficacy to selected 3 kinds of indicator viruses for the 2 kinds of techniques such as line irradiation 25kgy.

1. verification sample: take from continuous three batch samples of inactivation technology back in production process, preserve in 4 DEG C.

2. indicator virus selects:

(1) Pseudorabies virus (prv): belong to herpetoviridae, be that double-strand dna has togavirus.To liposoluble such as ether, chloroforms Agent, the sensitivity such as formalin and ultraviolet irradiation, is stronger one kind of resistance in herpesviral.For traditional Chinese medicines prison note [2002] The hbv indicator virus of No. 160 literary composition regulations.

(2) Xin get Bi Shi virus (sindbis): belong to Alphaherpesvirinae, ball-type, is that rna has togavirus.To physical and chemical factor Resistance relatively low.Hcv indicator virus for traditional Chinese medicines [2002] No. 160 literary composition regulations of prison note.

(3) pig parvoviral (ppv): belong to Parvoviridae, be small dna no togavirus, 18-24nm.Due to no capsule Film is very strong to physical and chemical factor resistance.Non-fat coating parvovirus b19 instruction disease for traditional Chinese medicines [2002] No. 160 literary composition regulations of prison note Poison.

3. inactivation of virus/removal verification step:

(1) 0.5% glutaraldehyde solution 50 minutes inactivation technologies of immersion:

Allogenic material sample is put in the blake bottle after 50ml sterilization, is separately added into 0.5% glutaraldehyde solution 18ml, 2ml indicator virus solution (10%).Sampled respectively at 0,5,10,20,30,50 minutes, according to the knot of cell toxicity test Fruit maintaining liquid does detection virus residual titre after suitable dilution, and the continuation inactivation that both can terminate 0.5% glutaraldehyde solution is made With can eliminate the toxicity to detection cell for 0.5% glutaraldehyde solution again.It is multiple that every kind of sample does three batch weight.

(2) 60Co-γ rays irradiation 25kgy inactivation technology:

Allogenic material sample is put in the blake bottle after 50ml sterilization, is separately added into 20ml indicator virus solution straight Connect immersion.Taking-up 1ml does and is suitably diluted to predose correspondence immediately, and remaining sample is 60Co-γ rays irradiation 25kgy.Irradiation Sample is taken to measure virus residual titre with predose sample with same dilution factor after end.It is multiple that every kind of sample does three batch weight.

4. virus activity titration method:

(1) cytotoxicity detection:

In order to determine 0.5% glutaraldehyde solution inactivation after sample determination virus residual titre when initial dilution it is necessary to First do the toxicity test to detection cell for the 0.5% glutaraldehyde solution difference dilution factor, just can carry out virus activity titration.But it is logical Cross the continuation deactivation that dilution terminates 0.5% glutaraldehyde solution.Mtt colorimetric method is used in cytotoxicity detection.

(2) virus activity titration:

From the sensitive vero and pk-15 cell line of virus, using Microdose cytopathic effect assay.By in above-mentioned each group different when Between the sample that taken according to cytotoxicity testing result, determine the initial dilution of sample, then do ten times be serially diluted after, immediately Add in 96 orifice plates having inoculated detection cell, each dilution factor does 8 holes to be repeated, and puts 37 DEG C, is incubated in 5%co2 incubator.With Vero cell detection prv and sindbis virus incubation 72 hours, light Microscopic observation simultaneously records cytopathy (cpe) situation.Due to The cytopathogenic effect of ppv is weaker, so the fluorescence antibody dye of specific anti-ppv on the basis of cytopathy judges, need to be increased Color in fluorescence microscopy Microscopic observation.The cell being infected by ppv is visible beautiful emerald green glimmering in the case of occurring without pathology Light spot, the method can significantly improve specificity and the sensitivity of ppv detection.Virus titer is pressed karberShi method and is calculated.

5. result judgement:

Viral reduction amount >=4lgtcid50/ml, judges that this viral inaction steps is effective.

6. cell blind passage three generations:

Every batch of equal high concentration of sample after the inactivation of various virus inactivation technologies adds in passage cell, does cell blind passage 3 In generation, see whether that cytopathy occurs.In the overall process in cell blind passage 3 generation, cytopathy and is the positive in any time (+), illustrate virus not by complete inactivation；Do not occur cytopathy be negative (-), illustrate virus by complete inactivation.

7. 0.5% glutaraldehyde solution is to cytotoxicity testing result:

7.1 0.5% glutaraldehyde solutions the results are shown in Table 1 to the toxicity detection of two kinds of detection cells

The impact to detection cell growth for table 1 0.5% glutaraldehyde solution

8. 0.5% glutaraldehyde solution processes different time to three kinds of model virus inactivations in " allogenic material sample " The observation of effect:

8.1 0.5% glutaraldehyde solutions process the inactivating efficacy to prv virus for the different time: (as shown in table 2, table 3)

Table 2 0.5% glutaraldehyde solution processes the inactivating efficacy to prv virus for the different time

Table 3 0.5% glutaraldehyde solution soaks blind passage result after 50 minutes inactivation prv virus

Sample number	A blind passage generation	Blind passage two generation	Blind passage three generations
				1	(-)	(-)	(-)
2	(-)	(-)	(-)
				3	(-)	(-)	(-)
Cell controls	(-)	(-)	(-)

8.2 0.5% glutaraldehyde solutions soak the inactivating efficacy to sindbis virus for the different time: (as table 4, table 5 institute Show)

Table 4 0.5% glutaraldehyde solution soaks the inactivating efficacy to sindbis virus for the different time

Table 5 0.5% glutaraldehyde solution soaks blind passage result after 50 minutes inactivation sindbis virus

8.3 0.5% glutaraldehyde solutions soak the inactivating efficacy to ppv virus for the different time: (as shown in table 6, table 7)

Table 6 0.5% glutaraldehyde solution soaks the inactivating efficacy to ppv virus for the different time

Table 7 0.5% glutaraldehyde solution soaks blind passage result after 50 minutes inactivation ppv virus

9. 60Co-γ rays irradiation 25kgy process " allogenic material sample " is to three kinds of model virus inactivating efficacies Observe:

9.1 60Co-γ rays irradiation 25kgy process " allogenic material sample " are to prv inactivation of virus effect: (as table 8th, shown in 9)

Table 8 60Co-γ rays irradiation 25kgy treatment of allogeneic material sample is to prv inactivation of virus effect

Blind passage result after table 9 60Co-γ rays irradiation 25kgy treatment of allogeneic material sample inactivation prv

Sample number	A blind passage generation	Blind passage two generation	Blind passage three generations
				1	(-)	(-)	(-)
2	(-)	(-)	(-)
				3	(-)	(-)	(-)

Cell controls

(-)

9.2 60Co-γ rays irradiation 25kgy process " allogenic material sample " are to sindbis inactivation of virus effect: (as shown in table 10,11)

Table 10 60Co-γ rays irradiation 25kgy processes specimen material to sindbis inactivation of virus effect

Table 11 60Co-γ rays irradiation 25kgy processes blind passage result after specimen material inactivation sindbis

9.3 60Co-γ rays irradiation 25kgy process " allogenic material sample " are to ppv inactivation of virus effect: (as table 12nd, shown in 13)

Table 12 60Co-γ rays irradiation 25kgy processes specimen material to ppv inactivation of virus effect

Table 13 60Co-γ rays irradiation 25kgy processes blind passage result after specimen material inactivation ppv

10. conclusion:

Checking test result shows, two in the production of product prepared by the present invention virus inactivation technology all can effectively go out Three kinds of added model virus of work.0.5% glutaraldehyde solution soaks 50 minutes technique, processes and gets final product complete inactivation in 5 minutes Prv, sindbis virus.Because ppv is no coating parvovirus, it is substantially free of albumen, extremely strong to chemical factors patience, need Processing within 30 minutes could this virus of complete inactivation.Viral reduction amount is respectively as follows: prv virus >=6.0-6.13lgtcid₅₀； Sindbis virus >=5.13-5.5lgtcid₅₀；Ppv virus >=4.75-4.88lgtcid₅₀.0.5% glutaraldehyde solution soaks 50 What minute was processed adds the detection of cell blind passage three generations's flaviviridae viral containing viral sample.60Co-γ rays 25kgy irradiation technique can go out Live prv virus >=6.38-7.13lgtcid₅₀；Sindbis virus >=5.75-5.88lgtcid₅₀；Ppv virus >=6.0- 6.25lgtcid₅₀.Three kinds of viral sample of irradiation 25kgy detect through cell blind passage three generations's flaviviridae viral.

Eat medicine prison note [2008] 7 and the regulation of traditional Chinese medicines prison [2002] No. 160 files of note according to state, in every step inactivation technology In, viral reduction amount reaches more than 4logs it is believed that inactivation of viruses method is effective.And each technique can to viral deactivation To be superimposed.Viral reduction amount is bigger, and the virus-free detection of blind passage three generations, illustrates that inactivation technology effect is better, the security of product Higher.This result of the test shows, two techniques of checking, and viral reduction amount is all higher than 4logs, if two kinds of technique superpositions, three kinds The reduction amount of indicator virus all reaches more than 10logs.

So, 0.5% glutaraldehyde solution in preparation method of the present invention soaks 50 minutes, 60Co-γ rays 25kgy Irradiation two-strain inactivation technology can ensure that the Viral safety of such product.

3rd, the immunogenicity checking to the human-body biological dressing made by embodiment of the present invention is as follows:

Checking foundation: animal derived medical device product registration declaration material writes guideline

Verifying purpose: by implanting to rabbit 2 weeks after dressing of the present invention, 4 weeks, 12 weeks serum antibody igg, iga, Igm situation of change, evaluates the immunogenicity of product prepared by the present invention.

1. laboratory sample: the biological human biological dressing producing according to preparation method provided in an embodiment of the present invention, produce Lot number: 1 and 2, it is placed in 4 DEG C of refrigerators in buffer solution and deposit.

2. animal used as test, reagent and instrument and equipment

Animal used as test: white big ear rabbit 60, body weight 2.05 ± 0.21kg, hero is female not to be limited.

Main agents: rabbit igg, iga, igm elisa kit, two kinds of specifications of t96, t48；Immunologic adjuvant, 10ml/ props up； 5% amobarbital parenteral solution；Gentamicine sulphate injection, 80,000 iu/2ml.

Key instrument equipment: agitator；Centrifuge；Refrigerator；Electro-heating standing-temperature cultivator；ELIASA.

3. blood specimen collection

Before experiment and 2 weeks after test, gather blood sample within 4 weeks, 12 weeks.Gather non-anti-freezing with asepsis injector through rabbit arteria auricularis Blood 1ml, puts 1h at room temperature, after solidification, puts 4 DEG C of refrigerator overnight and separates out serum, be centrifuged 10min under 3500rpm, suction out blood Clearly, it is sub-packed in ep pipe, after mark cleaning, be stored in -80 DEG C of refrigerators.After the completion of all taking a blood sample after 12 weeks, unification carries out antibody Igg, iga, igm horizontal detection.

4. the preparation of biological human biological dressing leachate

Human-body biological dressing is shredded by super-clean bench, is placed in mixer, add the phosphoric acid buffer of sterilized ph7.0 Liquid, stirs into homogenate, and in experiment, apparatus used is all processed through autoclaving.Homogenate is placed in 37 DEG C of electro-heating standing-temperature cultivators and placed After night, under 12000rpm, it is centrifuged 10min, take supernatant through 0.22 μm of sterilizing filter filtration sterilization.Put in 4 DEG C of refrigerators and deposit Put.The preparation in 1 day before use of human-body biological dressing leachate.

5. experiment packet and process

Experimental rabbit (60) is divided into four groups: high dose group (implantation human-body biological dressing group), low dose group (leachate Group), negative control group, positive controls (booster immunization group).Every group 15.

High dose group (implantation human-body biological dressing group): rabbit 5% anaesthetized with pentobarbital, dosage is 0.5ml/ Kg, slowly injects through ear edge artery.After the depilation of rabbit back local skin, sterilization, in incision of skin, human-body biological dressing is implanted In skin, stitching processing.

Low dose group (leachate group): take human-body biological dressing leachate 0.7ml, divide at 6 points and be injected in rabbit vertebra both sides Subcutaneous, every injection about 0.12ml.

Negative control group: modus operandi is identical with high dose group, but do not implant human-body biological dressing.

Positive controls (booster immunization group):

Human-body biological dressing leachate and the preparation of adjuvant mixed liquor: by human-body biological dressing leachate and adjuvant equal-volume Mixing, mixed liquor is sucked respectively in the sterilizing glass syringe of two 100ml, two syringes are about the aseptic of 10cm with one and mould Expects pipe connects, and push-and-pull two syringe makes liquid blending respectively, forms water-in-oil emulsion.Emulsion is dripped one and drops on the water surface, emulsion Tear drop is kept completely and not disperse as qualified.Human-body biological dressing leachate and adjuvant mixed liquor are prepared before use.

With adding the human-body biological dressing leachate immunizing rabbit of adjuvant: take human-body biological dressing leachate to mix with adjuvant Liquid 1.5ml, points of 6 points subcutaneous, the every injection about 0.25ml that are injected in rabbit vertebra both sides.After 2 weeks, then the human body with same dose With adjuvant mixed liquor booster immunization once, method is the same for biological dressing leachate.

For preventing POI, the rabbit (high dose group and negative control group) performed the operation was on the operation same day and postoperative 3 Its intramuscular injection gentamicine sulphate injection, 40,000 units/sky.

2 weeks, 4 weeks, the blood sample of 12 weeks after collection experiment.In experimentation, observe animal wound healing situation, have or not redness Ooze out situation；Situations such as animal activity, feed, drinking-water and animal survival condition.

6.elisa detects

Serum antibody igg, iga, igm horizontal detection operates according to kit specification.Assay method is as follows:

(1) dilution of standard items and sample-adding: according to the Serum antibody concentrations scope that preliminary experiment obtains, standard items are carried out dilute Release.Igg standard items are diluted to five concentration point of 1.5,3,6,12,18 μ g/ml, iga, igm standard items are all diluted to 0.75,1.5, 3rd, five concentration point of 6,9 μ g/ml.50 μ l standard dilutions are added enzyme mark to be coated in plate gauge orifice.

(2) be loaded: set respectively blank well (blank control wells are not added with sample and enzyme marking reagent, and remaining each step operation is identical), Testing sample hole.It is coated in testing sample hole on plate in enzyme mark and first adds sample diluting liquid 40 μ l, then add testing sample 10 μ l again (the final dilution factor of sample is 5 times).

(3) incubate: with the rearmounted 37 DEG C of incubation 30min of shrouding film shrouding.

(4) prepare cleaning solution: by 30 times of concentrated cleaning solutions with standby after the dilution of 30 times of distilled water.

(5) wash: carefully take shrouding film off, discard liquid, dry, cleaning solution is filled it up with every hole, discards, weight after standing 30s Multiple 5 times, pat dry.

(6) enzyme-added: every hole adds enzyme marking reagent 50 μ l, except blank well.

(7) incubate: operation is with (3).

(8) wash: operation is with (5).

(9) develop the color: every hole is initially charged developer a50 μ l and adds developer b50 μ l, gently concussion mixes, 37 DEG C of lucifuges Colour developing 15min.

(10) terminate: every hole adds terminate liquid 50 μ l, terminating reaction.

(11) measure: with blank well zeroing, 450nm wavelength sequentially measures the absorbance in each hole.Mensure should add terminate liquid Carry out within 15min afterwards.

Three wells detects, result of averaging is judged.

7. statistical analysis

Statistical analysis are carried out using spss16.0 software.Measurement data is usedRepresent, between each group and different sampling The contrast of the antibody test result of time adopt one-way analysis of variance check, rate relatively adopt χ²Detection.All statistics push away Break all using two-sided test, the inspection level with statistical significance is set to p < 0.05.

8. immunogenicity experiments result

The ordinary circumstance of 8.1 animals used as test

The rabbit performed the operation visible activity in a day after surgery reduces, and feed reduces, and recovers normal, no wound infection later Occur, the no red and swollen diffusate of wound, animal wound healing is good, and obvious abnormal response in No operation rabbit.Each group is dead Number of animals is shown in Table 14.

Table 14 each group dead animal number

χ is carried out to each group mortality of animals²Detection, obtains χ²=0.373, p=0.946 > 0.05, show that between each group, rabbit is dead Rate of dying no difference of science of statistics, the death of rabbit is unrelated with human-body biological dressing implantation.

Contrast between the change of 8.2 Serum Antibodies of Rabbits and each group

8.2.1 Serum Antibodies of Rabbits igg, iga, igm level

Serum Antibodies of Rabbits igg, iga, igm testing result is listed in table 15.

Table 15 Serum Antibodies of Rabbits igg, iga, igm level (μ g/ml)

8.2.2 the change of different time Serum Antibodies of Rabbits after processing

After process, Fig. 3-5 is shown in the change of different time Serum Antibodies of Rabbits.It can be seen that each group rabbit anteserum Antibody horizontal all raises in second week, has declined in 4th week, but has remained above the foundation level before experiment, and serum resists within the 12nd week Body decreases to substantially the foundation level before experiment.

Statistical test is carried out to different time points antibody level of serum, result lists table 16-19 in.From statistical analysis knot Fruit as can be seen that high dose group and positive controls second week igg, iga, igm level is above testing front level after treatment, There is the meaning of significance,statistical；After low dose group is processed, second week iga and igm level are higher than level before experiment, and igg With the level contrast no difference of science of statistics before experiment；Negative control group after treatment second week igg level be also higher than experiment before Level, may cause slight immune response relevant with operation wound；High dose group and positive controls 4th week iga after treatment Still it is significantly higher than the level before experiment with igm level；Each group all substantially returns to the level before testing after treatment on the 12nd week, with Comparative result no difference of science of statistics before experiment.

The results of statistical analysis of table 16 negative control group different time points antibody horizontal

The results of statistical analysis of table 17 high dose group different time points antibody horizontal

The results of statistical analysis of table 18 low dose group different time points antibody horizontal

The results of statistical analysis of table 19 positive controls different time points antibody horizontal

8.2.3 each group Serum Antibodies of Rabbits level contrast

The 2nd week difference is more apparent after treatment for each group Serum Antibodies of Rabbits level (Fig. 6-9), the 2nd week each group rabbit anteserum Antibody horizontal is shown in Fig. 7, it can be seen that serum antibody igg, iga, igm level is: positive controls > high dose group > Low dose group > negative control group.Statistical test is carried out to each group Serum Antibodies of Rabbits level, result is listed in table 20-22.From table In as can be seen that process after the 2nd week high dose group igg, iga, igm level be above negative control group, the 4th week iga and igm water Put down and remain above negative control group, result has statistical significance, illustrate that human-body biological dressing implantation can cause rabbit to a certain degree Immune response, but antibody is still in reduced levels, and igg, iga, igm highest level is only respectively 25.93,10.16,9.98 μ g/ml.Using adjuvant booster immunization, with high dose group contrast, have no that antibody horizontal significantly improves, show human-body biological dressing warp After going antigen to process, immunogenicity is very faint, even if adopting adjuvant booster immunization, is also not result in obvious immune response.

The results of statistical analysis of table 20 each group rabbit anteserum igg level

The results of statistical analysis of table 21 each group rabbit anteserum iga level

The results of statistical analysis of table 22 each group rabbit anteserum igm level

9. conclusion

After 9.1 implantation human-body biological dressing, obvious abnormal response in rabbit, shows that human-body biological dressing implantation is Safety；

9.2 serum antibodies reach highest level in 2 weeks after human-body biological dressing implantation, decrease after 4 weeks, 12 Zhou Shiyi Foundation level before experiment；

After 9.3 human-body biological dressing implantation, the 2nd week antibody level of serum is higher than negative control group, the 4th week iga and igm water Put down and remain above negative control group, after human-body biological dressing implantation is described, rabbit a certain degree of immune response can be caused 2-4 week, but Antibody is still in relatively low expression, and igg, iga, igm highest level is only respectively 25.93,10.16,9.98 μ g/ml；Adopt Use adjuvant booster immunization, also have no that antibody horizontal significantly improves, show that the immunogenicity of human-body biological dressing is very weak.

4th, to the human-body biological casting product clinical testing one made by embodiment of the present invention:

Clinical physical data (selection of disease, total cases and case): this group burns (wound) sick and wounded people 30, wherein man 21 Example, female 9；Age 2-58 year；Minimum area 1%, maximum area 5%；Totally 36 surface of a wound, the wherein fresh shallow ii ° of surface of a wound 12, The fresh deep ii ° of surface of a wound 6, residual wound 6, cut (cutting) scab skin grafted woundses 6, skin donor site 6.This group case is same Qimen Examine and inpatient.

Clinical testing procedure: this group experimental technique is carried out by clinical trial protocol, test group uses prepared by the present invention Human-body biological dressing covers all kinds of burnings (wound) surface of a wound, and control group selects Petroleum gauze or other biological dressing.Test group with right Same position according to the same patient of group selection. or the surface of a wound of different parts carries out comparitive study.

The evaluation criterion taken or/and statistical method: the ratio of surface of a wound routine healing time and test group healing time Relatively. processed from t test statistics method, as the foundation of the human-body biological dressing curative effect evaluated prepared by the present invention.Cover Cut the autologous little skin graft of (cutting) scab or the wear debris emission surface of a wound, gradually drying comes off for human-body biological dressing used above in postoperative 5 weeks, Surface of a wound closing person is considered as excellent；It is bad that less than 4 weeks persons are considered as difference.

Clinical test results: this group 30,36 surface of a wound, only 1 children's surface of a wound loosely re-replaces because fixing.Newly The healing in general 8 10 days of the fresh shallow ii ° of surface of a wound, average shortening 2-4 days；The healing in general 13-17 days of the deep ii ° of surface of a wound, averagely shortens 3 5 days；Surface of a wound NIP reacts.Use in granulation wound, chronic ulcer, bedsore, removable slough, purification, the cleaning surface of a wound, Beneficial to fresh granulation growth, wound healing or for skin-grafting repair cultivated good wound base.Boundary lid is cut (cutting) scab particulate and is planted The skin surface of a wound, human-body biological dressing mummification used above in postoperative 5 weeks comes off, and improves healing quality.The surface of a wound is checked after 2 months, no bright Aobvious scar proliferation phenomenon；And not using the human-body biological dressing person prepared by the present invention, have scar proliferation phenomenon.Laboratory examination: Blood, routine urinalysis and Liver and kidney function are without exception, and thin rice seedling culture no grows；Clinical practice has no toxic and side effect.

Clinical trial result is analyzed: the human-body biological dressing prepared by the present invention is effectively protected the surface of a wound, shortens more Ask during conjunction: for the epidermis avulsion surface of a wound, " seamless " can be formed and immediately cover；Especially dermal tissue face has carried out removing antigen Technical finesse, reduces antigen dose, digs and upper use cutting the transplanting of (cutting) scab particulate, adhesive force is strong, histocompatbility is good, prolongs Grow the time of flap coverage, improve the outward appearance after wound healing and function.

Clinical testing conclusion: the human-body biological casting product prepared by the present invention is it is adaptable to cover all kinds of burnings (wound) wound wound Face and the Micrograiting surface of a wound, are effectively protected the surface of a wound, block fourth bacterium and invade eight, shorten healing time；Skin donor site extremely lacks Patient, first covering human-body biological dressing of the present invention can be the guarantee of offer time of next time making skin graft.Purification, cleaning is had to burst The effect of ulcer, bedsore and granulation wound, promotes fresh granulation tissue growth, and the surface of a wound can have been cultivated good with self-healing or for skin-grafting reparation Good epithelial cell growth environment, is a kind of surface of a wound external application human-body biological dressing of novel concept being worthy to be popularized.

5th, to the human-body biological casting product clinical testing two made by embodiment of the present invention:

Clinical physical data (selection of disease, total cases and case): this group all select the same time period outpatient service and Inpatient 32: man 18, female 14；Age 1-36 year；Totally 42 surface of a wound: shallow ii ° of example (8 surface of a wound), deep ii ° of example (8 The surface of a wound), large area ° 5 (15 surface of a wound)；Granulation wound 4 (4 surface of a wound), chronic ulcer 2 (2 surface of a wound), bedsore 2 Example (2 surface of a wound)；Fresh wound and wound surface 3 (3 surface of a wound).This group use minimum area 6cm × 4cm, maximum area 50cm × 40cm.

Clinical testing procedure (includes control group setting): 1, test group and control group case adopt random packet: test group Cover all kinds of burnings (wound) using the embodiment of the present invention made human-body biological dressing and hinder the surface of a wound, and control group covering material selects all scholars Woods oil yarn or biological dressing 2, select consubstantiality or the approximate surface of a wound of same site tissue damage as the ratio of test group and control group curative effect Relatively.

The evaluation criterion taken or/and statistical method: 1, with all kinds of surface of a wound routine healing time as standard, with test The time of group more platform compares, and the statistical method from t inspection is processed, as evaluation human-body biological dressing of the present invention contracting The foundation of short wound healing time；2nd, cover the Micrograiting surface of a wound, come off within postoperative more than 5 weeks, it is up to standard qualified to be considered as；Less than 4 weeks Come off for unqualified.

Clinical test results: this group 32,42 surface of a wound, wherein 2 surface of a wound loosely re-replace because fixing.Shallow ii ° The surface of a wound is healed platform for 7-9 days, shortens 3-5 days；The healing in 2 weeks about of the deep ii ° of surface of a wound, shortens 5-7 days；Have no inflammatory reaction.Granulation wound, Use in chronic ulcer, bedsore, removable slough, purification, the cleaning surface of a wound, beneficial to the growth of fresh granulation, promote the surface of a wound to heal Close or cultivated good wound base for skin-grafting.Blood, routine urinalysis and Liver and kidney function no abnormality seen, Bacteria Culture no grows, and has no poison Side effect.(cutting) scab Micrograiting surface of a wound is cut in covering °, comes off within postoperative more than 5 weeks, improves healing quality.

Clinical trial result is analyzed: the human-body biological dressing prepared by the present invention has the advantages that allograft skin flap coverage, Create good internal environment for wound repair, shorten wound healing time.Additionally, the human-body biological prepared by the present invention The dermal tissue face of dressing, the technical finesse through past antigen, there is the effect of a thin layer extracellular matrix " support ", improve The outward appearance of the surface of a wound and function after healing.

Clinical testing conclusion: the human-body biological dressing prepared by the present invention, be applied to cover all kinds of burnings (wound) hinder the surface of a wound and The Micrograiting surface of a wound, has good covering effect, mitigates surface of a wound pain, promotes epithelial growth, shorten healing time, reduce scar Trace is formed；In addition, cover ulcer, bedsore and granulation wound and, can purify, clean the surface of a wound, promote fresh granulation growth, beneficial to wound Heal or repair for skin-grafting and cultivated good wound base in face.It is a kind of surface of a wound external application human-body biological of novel concept being worthy to be popularized Dressing.

Above-described only the preferred embodiments of the present invention, the explanation of should be understood that above example is to use In help understand the method for the present invention and its core concept, the protection domain being not intended to limit the present invention, all the present invention's Any modification of being made within thought and principle, equivalent etc., should be included within the scope of the present invention.

Claims

1. a kind of semi-finished product of the human-body biological dressing for covering the burn trauma surface of a wound are it is characterised in that its preparation method includes The following step carrying out in order:

Step [1] is 0.25%-3%'s in the mass fraction that the ratio that allograft skin every square centimeter takes 0.5-3ml takes respective amount Glutaraldehyde solution, allograft skin is put in taken glutaraldehyde solution, soaks 15-60 minute；Change glutaraldehyde solution, continue to soak 15-30 minute；Take out allograft skin, in the mass fraction that the ratio that allograft skin every square centimeter takes 0.5-3ml takes respective amount be The sodium chloride solution of 0.5%-1.5%, allograft skin is put in taken sodium chloride solution, soaks 30-120 minute, takes out allosome Skin obtains semi-finished product a；

Semi-finished product a is split by step [2] by required specification, with taking skin drum that the semi-finished product a after segmentation is carried out anti-take, removes The adipose tissue of remaining, obtains semi-finished product b；

Step [3] takes the ratio of 0.5-3ml to take the purified water of respective amount in semi-finished product b every square centimeter, by semi-finished product b and being taken Purified water is placed in same container, is positioned over shaking table concussion 15-60 minute, changes purified water, is again placed in shaking table concussion 15-60 Minute, repeat above step, until when the ph value of the purified water displacing is between 5.5-6.5, discarding purified water, obtaining semi-finished product c；

It is 0.05%-0.3% that step [4] takes the ratio of 0.5-3ml to take the mass fraction of respective amount in semi-finished product c every square centimeter Polyoxyethylene sorbitan monoleate solution, semi-finished product c and taken polyoxyethylene sorbitan monoleate solution are placed in same container, be positioned over shaking table concussion 15-60 minute, changes polyoxyethylene sorbitan monoleate solution, is again placed in shaking table concussion 15-60 minute；Repeat above step, until displacement When the ph value of the polyoxyethylene sorbitan monoleate solution going out is between 6.0-7.0, discard polyoxyethylene sorbitan monoleate solution, obtain semi-finished product d；

Step [5] takes the ratio of 0.5-3ml to take the purified water of respective amount in semi-finished product d every square centimeter, by semi-finished product d and being taken Purified water is placed in same container, is positioned over shaking table concussion 15-60 minute, changes purified water, is again placed in shaking table concussion 15-60 Minute, repeat above step, until when the ph value of the purified water displacing is between 5.5-6.5, discarding purified water, obtaining semi-finished product e；

Step [6] takes the ratio of 0.5-3ml to take the Freamine Ⅲ of respective amount in semi-finished product e every square centimeter, by semi-finished product e and Taken Freamine Ⅲ is placed in same container, puts shaking table concussion 15-60 minute, then stands 15-30 minute, discard amino acid Solution, obtains semi-finished product f；Wherein, described amino acid is glutamic acid, alanine, glycine, asparatate one of work as or number Kind；

Step [7] takes the ratio of 0.5-3ml to take the purified water of respective amount in semi-finished product f every square centimeter, by semi-finished product f and institute Take purified water to be placed in same container, be positioned over shaking table concussion 15-60 minute, change purified water, be again placed in shaking table concussion 15- 60 minutes, repeating above step, until when the ph value of the purified water displacing becomes between 6.0-7.0, discarding purified water, obtaining half Finished product g；

Step [8] takes the ratio of 0.5-3ml to take the pbs buffer solution of respective amount in semi-finished product g every square centimeter, by semi-finished product g and Taken pbs buffer solution is placed in same container, is positioned over shaking table concussion 15-60 minute, then stands 15-30 minute, discard institute The pbs buffer solution using, obtains semi-finished product h；Wherein, the ph value of described pbs buffer solution is 7.0-8.0；

Foreign matters all in the corium face of semi-finished product h, epidermis side are removed clean, if there being a diameter of more than 1 centimetre by step [9] Hole, then sewed on hole with medical suture, if shape is irregular, by its trimmed, obtains semi-finished product i；

It is 0.5%-1.5% that step [10] takes the ratio of 0.5-3ml to take the mass fraction of respective amount in semi-finished product i every square centimeter Sodium chloride solution, then semi-finished product i and taken sodium chloride solution are placed in same container, are positioned over shaking table and shake 15-60 Minute, change sodium chloride solution, be again placed in shaking table concussion 15-60 minute.Repeat above step, until the sodium chloride displacing When the ph value of solution is between 6.5-7.5, take out semi-finished product i；The ratio of 1ml is taken to take accordingly in every 1-3 square centimeter semi-finished product i The mass fraction of amount is the sodium chloride solution of 0.5%-1.5%, and semi-finished product i and taken sodium chloride solution are placed in same container In, it is standing and soak for 15-60 minute, discards solution, obtain final semi-finished product j.

2. a kind of human-body biological dressing for covering the burn trauma surface of a wound is it is characterised in that its preparation method includes entering in order The following step of row: step [1] glutaraldehyde solution immersion treatment, step [2] segmentation, step [3] purified water immersion treatment, step [4] polyoxyethylene sorbitan monoleate solution immersion treatment, step [5] purified water immersion treatment, step [6] Freamine Ⅲ immersion treatment, step Suddenly [7] purified water immersion treatment, step [8] pbs buffer solution immersion treatment, step [9] is repaired and finishing, step [10] sodium chloride Solution immersion treatment and step [11] packaging and irradiation；

Wherein, step [1] is specially and in the mass fraction that the ratio that allograft skin every square centimeter takes 0.5-3ml takes respective amount is The glutaraldehyde solution of 0.25%-3%, allograft skin is put in taken glutaraldehyde solution, soaks 15-60 minute；Change glutaraldehyde Solution, continues to soak 15-30 minute；Take out allograft skin, take respective amount in the ratio that allograft skin every square centimeter takes 0.5-3ml Mass fraction is the sodium chloride solution of 0.5%-1.5%, and allograft skin is put in taken sodium chloride solution, soaks 30-120 and divides Clock, takes out allograft skin and obtains semi-finished product a；

Step [2] is specially to be split semi-finished product a by required specification, with taking skin drum that the semi-finished product a after segmentation is carried out instead Take, remove the adipose tissue of remaining, obtain semi-finished product b；

Step [3] obtains semi-finished product c by purified water immersion treatment；

Step [4] is specially and takes the ratio of 0.5-3ml to take the mass fraction of respective amount to be in semi-finished product c every square centimeter The polyoxyethylene sorbitan monoleate solution of 0.05%-0.3%, semi-finished product c and taken polyoxyethylene sorbitan monoleate solution are placed in same container, place Shake 15-60 minute in shaking table, change polyoxyethylene sorbitan monoleate solution, be again placed in shaking table concussion 15-60 minute, repeat above walking Suddenly, until when the ph value of the polyoxyethylene sorbitan monoleate solution displacing is between 6.0-7.0, discarding polyoxyethylene sorbitan monoleate solution, obtaining half one-tenth Product d；

Step [5] obtains semi-finished product e by purified water immersion treatment；

Step [6] is specially and takes the ratio of 0.5-3ml to take the Freamine Ⅲ of respective amount in semi-finished product e every square centimeter, will be partly Finished product e and taken Freamine Ⅲ are placed in same container, put shaking table concussion 15-60 minute, then stand 15-30 minute, abandon Deaminize acid solution, obtains semi-finished product f；Wherein, described amino acid be glutamic acid, alanine, glycine, in the middle of asparatate One or several；

Step [7] obtains semi-finished product g by purified water immersion treatment；

Step [8] is specially and takes the ratio of 0.5-3ml to take the pbs buffer solution of respective amount in semi-finished product g every square centimeter, will half one-tenth Product g and taken pbs buffer solution are placed in same container, are positioned over shaking table concussion 15-60 minute, then stand 15-30 minute, abandon Remove used pbs buffer solution, obtain semi-finished product h；Wherein, the ph value of described pbs buffer solution is 7.0-8.0；

Step [9] is repaired to semi-finished product h and is repaired to obtain semi-finished product i；

Step [10] is specially and takes the ratio of 0.5-3ml to take the mass fraction of respective amount to be in semi-finished product i every square centimeter The sodium chloride solution of 0.5%-1.5%, then semi-finished product i and taken sodium chloride solution is placed in same container, is positioned over and shakes Bed concussion 15-60 minute, changes sodium chloride solution, is again placed in shaking table concussion 15-60 minute；Repeat above step, until putting When the ph value of the sodium chloride solution swapping out is between 6.5-7.5, take out semi-finished product i；Take 1ml by every 1-3 square centimeter semi-finished product i Ratio take the sodium chloride solution that the mass fraction of respective amount is 0.5%-1.5%, semi-finished product i and taken sodium chloride solution are put In same container, it is standing and soak for 15-60 minute, discards solution, obtain semi-finished product j.

3. human-body biological dressing as claimed in claim 2 presses semi-finished product every square centimeter it is characterised in that step [3] is specially B takes the ratio of 0.5-3ml to take the purified water of respective amount, semi-finished product b and taken purified water is placed in same container, is positioned over and shakes Bed concussion 15-60 minute, changes purified water, being again placed in shaking table concussion 15-60 minute, repeating above step, until displacing Purified water ph value between 5.5-6.5 when, discard purified water, obtain semi-finished product c.

4. human-body biological dressing as claimed in claim 2 presses semi-finished product every square centimeter it is characterised in that step [5] is specially D takes the ratio of 0.5-3ml to take the purified water of respective amount, semi-finished product d and taken purified water is placed in same container, is positioned over and shakes Bed concussion 15-60 minute, changes purified water, being again placed in shaking table concussion 15-60 minute, repeating above step, until displacing Purified water ph value between 5.5-6.5 when, discard purified water, obtain semi-finished product e.

5. human-body biological dressing as claimed in claim 2 presses semi-finished product every square centimeter it is characterised in that step [7] is specially F takes the ratio of 0.5-3ml to take the purified water of respective amount, semi-finished product f and taken purified water is placed in same container, is positioned over and shakes Bed concussion 15-60 minute, changes purified water, being again placed in shaking table concussion 15-60 minute, repeating above step, until displacing Purified water ph value become between 6.0-7.0 when, discard purified water, obtain semi-finished product g.

6. human-body biological dressing as claimed in claim 2 it is characterised in that step [9] be specially by the corium face of semi-finished product h, In epidermis side, all foreign matters are removed totally, if there is a diameter of more than 1 centimetre of hole, are sewed on hole with medical suture, If shape is irregular, by its trimmed, obtain semi-finished product i.

7. a kind of human-body biological dressing for covering the burn trauma surface of a wound is it is characterised in that its preparation method is included in order The following step carrying out:

Step [1] is 0.25%-3%'s in the mass fraction that the ratio that allograft skin every square centimeter takes 0.5-3ml takes respective amount Glutaraldehyde solution, allograft skin is put in taken glutaraldehyde solution, soaks 15-60 minute；Change glutaraldehyde solution, continue to soak 15-30 minute；Take out allograft skin, in the mass fraction that the ratio that allograft skin every square centimeter takes 0.5-3ml takes respective amount be The sodium chloride solution of 0.5%-1.5%, allosome ratio is put in taken sodium chloride solution, soaks 30-120 minute, takes out allosome Skin obtains semi-finished product a；

Step [3] takes the ratio of 0.5-3ml to take the purified water of respective amount in semi-finished product b every square centimeter, by semi-finished product b and being taken Purified water is placed in same container, is positioned over shaking table concussion 15-60 minute, changes purified water, is again placed in shaking table concussion 15-60 Minute.Repeat above step, until when the ph value of the purified water displacing is between 5.5-6.5, discarding purified water, obtaining semi-finished product c；

It is 0.05%-0.3% that step [4] takes the ratio of 0.5-3ml to take the mass fraction of respective amount in semi-finished product c every square centimeter Polyoxyethylene sorbitan monoleate solution, semi-finished product c and taken polyoxyethylene sorbitan monoleate solution are placed in same container, be positioned over shaking table concussion 15-60 minute, changes polyoxyethylene sorbitan monoleate solution, is again placed in shaking table concussion 15-60 minute.Repeat above step, until displacement When the ph value of the polyoxyethylene sorbitan monoleate solution going out is between 6.0-7.0, discard polyoxyethylene sorbitan monoleate solution, obtain semi-finished product d；

Step [5] takes the ratio of 0.5-3ml to take the purified water of respective amount in semi-finished product d every square centimeter, by semi-finished product d and being taken Purified water is placed in same container, is positioned over shaking table concussion 15-60 minute, changes purified water, is again placed in shaking table concussion 15-60 Minute.Repeat above step, until when the ph value of the purified water displacing is between 5.5-6.5, discarding purified water, obtaining semi-finished product e；

Step [7] takes the ratio of 0.5-3ml to take the purified water of respective amount in semi-finished product f every square centimeter, by semi-finished product f and being taken Purified water is placed in same container, is positioned over shaking table concussion 15-60 minute, changes purified water, is again placed in shaking table concussion 15-60 Minute.Repeat above step, until when the ph value of the purified water displacing becomes between 6.0-7.0, discarding purified water, obtaining half one-tenth Product g；

It is 0.5%-1.5% that step [10] takes the ratio of 0.5-3ml to take the mass fraction of respective amount in semi-finished product i every square centimeter Sodium chloride solution, then semi-finished product i and taken sodium chloride solution are placed in same container, are positioned over shaking table and shake 15-60 Minute, change sodium chloride solution, be again placed in shaking table concussion 15-60 minute.Repeat above step, until the sodium chloride displacing When the ph value of solution is between 6.5-7.5, take out semi-finished product i；The ratio of 1ml is taken to take accordingly in every 1-3 square centimeter semi-finished product i The mass fraction of amount is the sodium chloride solution of 0.5%-1.5%, and semi-finished product i and taken sodium chloride solution are placed in same container In, it is standing and soak for 15-60 minute, discards solution, obtain semi-finished product j；

Semi-finished product j is loaded packing inside bag by step [11], paves, packing inside bag is sealed in packing inside bag, is then charged into outer Packaging bag is simultaneously sealed against, after through cobalt co⁶⁰Irradiation, final finished product k.

8. the biological dressing semi-finished product described in claim 1 or the human-body biological dressing described in any one of claim 2-7 are in system Application in standby extensive deep burn biological dressing.

9. the biological dressing semi-finished product described in claim 1 or the human-body biological dressing described in any one of claim 2-7 are in system Application in the covering biological dressing of standby all kinds of burns or wound and wound surface.

10. the biological dressing semi-finished product described in claim 1 or the human-body biological dressing described in any one of claim 2-7 are in system Application in the covering biological dressing of the standby Micrograiting surface of a wound.

Biological dressing semi-finished product described in 11. claims 1 or the human-body biological dressing described in any one of claim 2-7 are in system Application in the covering biological dressing of standby granulation wound or chronic ulcer and bedsore.

Biological dressing semi-finished product described in 12. claims 1 or the human-body biological dressing described in any one of claim 2-7 are in system Application in the covering biological dressing of standby ulcer, bedsore or granulation wound.

Biological dressing semi-finished product described in 13. claims 1 or the human-body biological dressing described in any one of claim 2-7 are in system The standby extensive deep burn that covers is cut scab or is cut the application in scab Micrograiting surface of a wound biological dressing.

14. biological dressing semi-finished product according to claim 1 or the human-body biological dressing of any one of claim 2-7, its It is characterised by: allograft skin described in step [1] derives from the skin that healthy human body contributes body, its thickness is 0.2-0.3 millimeter, institute The thickness difference between allograft skin is taken to be less than 0.1 millimeter.

15. biological dressing semi-finished product according to claim 1 or the human-body biological dressing of any one of claim 2-7, its It is characterised by: used in step [1], the mass fraction of glutaraldehyde solution is 0.5%, and used glutaraldehyde solution presses every square Centimetre allograft skin takes the ratio of 1ml to obtain, and first time soak time in glutaraldehyde solution for the described allograft skin is 50 minutes, the Secondary soak time is 25 minutes；Or, being the polyoxyethylene sorbitan monoleate solution that mass fraction is 0.2% used in step [4], institute The polyoxyethylene sorbitan monoleate solution using is obtained in the ratio that semi-finished product c every square centimeter takes 2ml, and semi-finished product c is in taken polysorbate In 80 solution, on shaking table, the time of concussion every time is 30 minutes.

16. biological dressing semi-finished product according to claim 1 or the human-body biological dressing of any one of claim 2-7, its It is characterised by: the Freamine Ⅲ described in step [6], amino acid contained therein is glutamic acid, alanine, glycine, sky One or several in the middle of L-aminobutanedioic acid, in the middle of every 1000 milliliters of Freamine Ⅲs, glutamic acid, alanine, glycine, lucid asparagus The content of one or several of propylhomoserin is respectively 0.1-3g respectively.

17. biological dressing semi-finished product according to claim 16 or human-body biological dressing it is characterised in that: in step [6] Amino acid contained by described Freamine Ⅲ is glutamic acid and glycine, in the middle of every 1000 milliliters of this Freamine Ⅲs, containing paddy Propylhomoserin 0.75g, 1.5g containing glycine.

18. biological dressing semi-finished product according to claim 1 or the human-body biological dressing of any one of claim 2-7, its It is characterised by: the pbs buffer solution ph value used in step [8] is 7.5.

19. biological dressing semi-finished product according to claim 1 or the human-body biological dressing of any one of claim 2-7, its It is characterised by: the distance between stitch when described in step [9] with medical suture slot apertures hole, should be controlled to be not more than 1 centimetre.

20. according to the human-body biological dressing of any one of claim 2-7 it is characterised in that: the irradiation of step [11] is specially warp Cobalt co⁶⁰Irradiation, dosage is: 20kgy≤irradiation dose≤30kgy.

21. human-body biological dressing according to claim 20 it is characterised in that: irradiation dose be equal to 25kgy.