CN103285420B - A method for production of a human body biological dressing for covering burn wounds - Google Patents
A method for production of a human body biological dressing for covering burn wounds Download PDFInfo
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Abstract
The present invention provides a method for production of a human body biological dressing for covering burn wounds. Through methods of biology, biochemistry, etc., antigen extraction for reducing immunogenicity is carried out to allograft, dermis and epidermis is retained, and the clinical effect of the obtained biological dressing is far better than the allograft. The method of the present invention includes the following sequential steps: immersion in a glutaraldehyde solution; segmentation; immersion in purified water; immersion in a polysorbate 80 solution; immersion in purified water; immersion in an amino acid solution; immersion in purified water; immersion in a PBS buffer solution; repair and recondition; immersion in a sodium chloride solution; and packaging and irradiation. The resulting product of the present invention can be used for purifying and cleaning wounds, promoting the growth of granulation tissues to cover granulation wounds, ulcers and bed sores, and shortening the healing time.
Description
Technical field
The present invention relates to a kind of preparation method of burn trauma wound surface cladding material, particularly a kind of allograft skin for covering burn trauma wound surface goes antigen to reduce the manufacture method of immunogenic human-body biological dressing.
Background technology
Burn is at ordinary times and skin trauma the most general during the war years, the various infringements that burnt degree causes all with the destruction of skin barrier effect with lose relevant.As: metabolism aggravation, temperature decline, excessive water scatter and disappear, 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 seem particularly important.People find that biomaterial contributes to the healing of skin trauma for a long time.Constantly go deep into along with to researching wound healing, people recognize gradually and use the object of dressing to be not only in order to flap coverage far away, and dressing also should be able to wound healing.1962, doctor Winter of London University delivered famous paper on Nature magazine, demonstrated the Healing of moist environment to wound surface.With close compared with pig partial thickness skin Wound Defect with polyethylene film, exposing aerial wound surface re-epithelialization probability increases by 50%, and this idea is all supported in a large amount of clinical practice both at home and abroad subsequently and basic research.
Nearly 4O has developed multiple temporary wound surface both at home and abroad and has covered dressing, and is widely used in clinical.The temporary wound surface of current Clinical practice covers dressing and mainly comprises: traditional dressing, synthetic dressing, natural biological dressing three major types.And the selection of burn wound's cladding material is burn treating, particularly important in a large-area burns trauma care ring.
Traditional dressing is following shortcoming as gauze, cotton pad etc. exist: wound surface 1. cannot be kept moistening, and wound healing postpones; 2. dressing fiber easily comes off, and causes foreign body reaction, impact healing; 3. wound granulation is organized and is easily grown in the mesh of dressing, can cause pain when changing dressings; 4., when dressing is soaked into, pathogen is easily passed through; When 5. changing dressings, the tissue of easy damaged new life; 6. workload of changing dressings is large.
In synthetic dressing, thin film class synthetic dressing is as too low in the water vapour permeability of the products such as Tegaderm, Dermafilm, Oprafiex, Opsite, easy hydrops, can bring out or increase the weight of to infect.Foam type synthetic dressing is as large in product holes such as Allevyn, Ixralo, and wound granulation is organized and easily grown into, and causes demoulding difficulty thus brings wound again, and being subject to germ contamination, easily leaving over detritus in wound surface.There is following shortcoming in aerosol type synthetic dressing such as the products such as Hydron, Aeroplast: adhesiveness and tensile strength poor; Moisture retention is poor, and wound surface water evaporation quantity is large; The water solublity of spraying film can make it be softened by wound exudate, and the retention time is short; Without infection control effect, infect for polluting under film easily occurs wound surface.Hydrogels dressing is as too poor in the mechanical performance of the products such as Duoderm, Comfeel, Restore, Intrasite, can cause dressing and wound separation, provide chance to the intrusion of antibacterial breeding after a large amount of absorption exudate because of the expansion of colloid.
Natural biological dressing comprises autologous skin, alloskin (abbreviation allograft skin), radiated pig skin (xenogenesis skin), amniotic membrane, cell-less corium ground substance, the dressing of collagen class etc.The optimal method of current flap coverage is autologous transplanting skin, but the Patients with Big Area Burn of burn surface area more than 50%, and autologous skin source seems wretched insufficiency.Skin allograft is that nearly 4O is proved one of comparatively effective skin substitute products, is a kind of more satisfactory wound-surface cover, has preferably skin barrier function.Poisture-penetrability, the adhesiveness of allograft skin are similar to autologous skin, can stop the loss of bacterial invasion and prevention wound surface water, electrolyte, protein and heat, and have good hemostasis and promote epithelization function.
Allograft skin has very long history in the application in burn field, has and alleviates patient suffering, the effect of wound healing in burn treating.Within 1966, Cochrane is the earliest by stored frozen allograft skin (cryopreserved allograft, CPA) be applied to burn wound to succeed, but the process of CPA and preservation quite trouble, and antigenicity is stronger, rejection is fast, cannot apply clinically.Allograft skin (g1ycerolpreservedallograft preserved by the glycerol of a kind of improvement after 1984, GPA) start to apply clinically, ratio skin-deep with CPA, GPA is easy to produce and preserve, antigenicity is lower, anti-infection ability is also comparatively strong, and time-to-live significant prolongation, obtains clinically and apply more widely.But current existing allograft skin exists preservation condition to be required high, antigenicity, has the clinical problem such as occupancy, pathogenic microorganism easy infection.The particularly obvious immunological rejection that causes due to its antigenicity existed, dissolving in general about 2 ~ 3 weeks, comes off, and wound surface is exposed, affects primary healing.In recent years along with the development of biotechnology, more novel allograft skin human-body biological dressing starts into people clinical.
Summary of the invention
In order to overcome the deficiencies in the prior art, the invention provides a kind of manufacture method of the human-body biological dressing for covering burn trauma wound surface, the present invention utilizes allograft skin as raw material, carry out going antigen to reduce immunogenicity process to allograft skin by the means such as biology, biochemistry, remain corium and epidermis, compared with GPA, its immunological rejection postpones, and applies and preserves more easy.Product not only reaches the object of early stage flap coverage, but also can wound healing, and clinical practice does not find obvious immunological rejection and systemic inflammatory response.
The technical solution adopted for the present invention to solve the technical problems is: a kind of manufacture method of the human-body biological dressing for covering burn trauma wound surface, comprises the following step carried out in order:
The mass fraction that step [1] gets respective amount in the ratio that every square centimeter of allograft skin gets 0.5 ~ 3ml is the glutaraldehyde solution of 0.25% ~ 3%, allograft skin is put into got glutaraldehyde solution, soaks 15 ~ 60 minutes; Change glutaraldehyde solution, continue immersion 15 ~ 30 minutes; Take out allograft skin, the mass fraction that the ratio of getting 0.5 ~ 3ml in every square centimeter of allograft skin gets respective amount is the sodium chloride solution of 0.5% ~ 1.5%, allosome ratio is put into got sodium chloride solution, soaks 30 ~ 120 minutes, and taking-up allograft skin obtains semi-finished product A;
Semi-finished product A is split by required specification by step [2], carries out anti-take, remove remaining fatty tissue, obtain semi-finished product B with bark fetching drum to the semi-finished product A after segmentation;
Step [3] gets the purified water of respective amount in the ratio that every square centimeter of semi-finished product B gets 0.5 ~ 3ml, semi-finished product B and purified water of getting are placed in same container, be positioned over shaking table concussion 15 ~ 60 minutes, change purified water, be again placed in shaking table concussion 15 ~ 60 minutes.Repeat above step, until when the pH value of the purified water displaced is between 5.5 ~ 6.5, discard purified water, obtain semi-finished product C;
Step [4] is the polyoxyethylene sorbitan monoleate solution of 0.05% ~ 0.3% in the mass fraction that the ratio that every square centimeter of semi-finished product C gets 0.5 ~ 3ml gets respective amount, semi-finished product C and polyoxyethylene sorbitan monoleate solution of getting are placed in same container, be positioned over shaking table concussion 15 ~ 60 minutes, change polyoxyethylene sorbitan monoleate solution, be again placed in shaking table concussion 15 ~ 60 minutes.Repeat above step, until when the pH value of the polyoxyethylene sorbitan monoleate solution displaced is between 6.0 ~ 7.0, discard polyoxyethylene sorbitan monoleate solution, obtain semi-finished product D;
Step [5] gets the purified water of respective amount in the ratio that every square centimeter of semi-finished product D gets 0.5 ~ 3ml, semi-finished product D and purified water of getting are placed in same container, be positioned over shaking table concussion 15 ~ 60 minutes, change purified water, be again placed in shaking table concussion 15 ~ 60 minutes.Repeat above step, until when the pH value of the purified water displaced is between 5.5 ~ 6.5, discard purified water, obtain semi-finished product E;
Step [6] gets the Freamine Ⅲ of respective amount in the ratio that every square centimeter of semi-finished product E gets 0.5 ~ 3ml, semi-finished product E and Freamine Ⅲ of getting are placed in same container, put shaking table concussion 15 ~ 60 minutes, then leave standstill 15 ~ 30 minutes, discard Freamine Ⅲ, obtain semi-finished product F;
Step [7] gets the purified water of respective amount in the ratio that every square centimeter of semi-finished product F gets 0.5 ~ 3ml, semi-finished product F and purified water of getting are placed in same container, be positioned over shaking table concussion 15 ~ 60 minutes, change purified water, be again placed in shaking table concussion 15 ~ 60 minutes.Repeat above step, until when the pH value of the purified water displaced becomes between 6.0 ~ 7.0, discard purified water, obtain semi-finished product G;
Step [8] gets the PBS buffer of respective amount in the ratio that every square centimeter of semi-finished product G gets 0.5 ~ 3ml, semi-finished product G and PBS buffer of getting are placed in same container, be positioned over shaking table concussion 15 ~ 60 minutes, then 15 ~ 30 minutes are left standstill, discard used PBS buffer, obtain semi-finished product H;
Step [9] is clean by all foreign bodies removals in the corium face of semi-finished product H, epidermis side, and the hole that diameter is more than 1 centimetre if exist, then sew on hole with medical suture, if shape irregularity, by its trimmed, obtains semi-finished product I;
The mass fraction that step [10] gets respective amount in the ratio that every square centimeter of semi-finished product I gets 0.5 ~ 3ml is the sodium chloride solution of 0.5% ~ 1.5%, then semi-finished product I and sodium chloride solution of getting are placed in same container, be positioned over shaking table concussion 15 ~ 60 minutes, change sodium chloride solution, be again placed in shaking table concussion 15 ~ 60 minutes.Repeat above step, until when the pH value of the sodium chloride solution displaced is between 6.5 ~ 7.5, take out semi-finished product I; The mass fraction that the ratio of getting 1ml in every 1 ~ 3 square centimeter of semi-finished product I gets respective amount is the sodium chloride solution of 0.5% ~ 1.5%, and semi-finished product I and sodium chloride solution of getting are placed in same container, leaves standstill immersion 15 ~ 60 minutes, discards solution, obtain semi-finished product J;
Semi-finished product J is loaded packing inside bag by step [11], paves in packing inside bag, is sealed by packing inside bag, then loads outer packaging bag and is sealed, finally by cobalt Co
60irradiation, final finished product K.
Allograft skin described in above-mentioned steps [1] derives from healthy human body and contributes the skin of body, and its thickness is 0.2 ~ 0.3 millimeter, the thickness difference of getting between allograft skin be no more than 0.1 millimeter.
Freamine Ⅲ described in above-mentioned steps [6], wherein contained aminoacid is one or several in the middle of glutamic acid, alanine, glycine, aspartic acid, in the middle of every 1000 milliliters of Freamine Ⅲs, the content of one or several of glutamic acid, alanine, glycine, aspartic acid is respectively 0.1 ~ 3g respectively.
The pH value of the PBS buffer described in above-mentioned steps [8] is 7.0 ~ 8.0.
When meeting hole with medical suture described in above-mentioned steps [9], the distance that should control between stitch is not more than 1 centimetre.
Through cobalt in above-mentioned steps [11]
the dosage of irradiation is: 20kGy≤irradiation dose≤30kGy.
Good effect of the present invention: the human-body biological dressing of gained of the present invention intactly remains three-dimensional configuration structure and the composition (growth framework) of extracellular matrix, can grow in order by inducing host cell, not only shorten wound healing time, also improve wound healing quality, decrease cicatrization; Its immunological rejection of deep burn mesh graft application products obtained therefrom of the present invention is low, effectively extend the cover time, avoid postoperative too early exposed wound surface, prevent traumatic infection and PD, for later by stages autologous skin-grafting creates condition, thus be more conducive to the application in extensive deep burn treatment.
Product of the present invention and wounds have the good compatibility, effectively can completely cut off wound surface to contact with the direct of the external world, serve temporary transient skin barrier effect, for wound healing provides a good repairing environment, be conducive to epithelial cell growth, shorten wound healing time and quality after improve wound healing.
Use the obtained product of the method for the invention to be applicable to the covering that all kinds of burning (wound) hinders wound surface, be applicable to light Ⅱ°, depthⅡ°, III ° cut (cutting) crust wound surface, Micrograiting wound surface, granulation wound and chronic ulcer and decubital ulcer covering treat.What this product had a skin complete removes antigen skin corium and epidermal area, while reducing immunological rejection, remain the corresponding function of skin corium and epidermal area, cover burning (wound) to hinder on wound surface and can play temporary transient skin barrier effect, can creating surviving of long period on base without obvious occupy-place, especially cover extensive deep burn and cut (cutting) crust Micrograiting wound surface, can primary healing improve wound repair quality.
Accompanying drawing explanation
Fig. 1 is the flow chart of the manufacture method of a kind of human-body biological dressing for covering burn trauma wound surface 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 tests, each group Serum Antibodies of Rabbits level compares.
Within after Fig. 7 uses human-body biological dressing process of the present invention 2 weeks, respectively organize Serum Antibodies of Rabbits level to compare.
Within after Fig. 8 uses human-body biological dressing process of the present invention 4 weeks, respectively organize Serum Antibodies of Rabbits level to compare.
Within after Fig. 9 uses human-body biological dressing process of the present invention 12 weeks, respectively organize Serum Antibodies of Rabbits level to compare.
Detailed description of the invention
Below in conjunction with accompanying drawing 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 manufacture method of the biological dressing for covering burn trauma wound surface, comprises the following step carried out in order:
Step [1] glutaraldehyde solution immersion treatment: (derive from the skin that healthy human body contributes body by every square centimeter of allograft skin, its thickness is 0.2 ~ 0.3 millimeter) ratio of the getting 1ml mass fraction of getting respective amount is the glutaraldehyde solution of 0.5%, allograft skin is put into got glutaraldehyde solution, soaks 50 minutes; Change glutaraldehyde solution, continue immersion 25 minutes; Take out allograft skin, the mass fraction that the ratio of getting 1ml in every square centimeter of alloskin gets respective amount is the sodium chloride solution of 0.9%, allograft skin is put into got sodium chloride solution, soaks 90 minutes, and taking-up allograft skin obtains semi-finished product A;
Step [2] is split: split by required specification by semi-finished product A, carries out anti-take, remove remaining fatty tissue, obtain semi-finished product B with bark fetching drum to the semi-finished product A after segmentation;
Step [3] purified water immersion treatment: the ratio of getting 2ml in every square centimeter of semi-finished product B gets the purified water of respective amount, semi-finished product B and purified water of getting are placed in same container, be positioned over shaking table and shake 30 minutes, change purified water, be again placed in shaking table and shake 30 minutes.Repeat above step, until when the pH value of the purified water displaced is between 5.5 ~ 6.5, discard purified water, obtain semi-finished product C;
Step [4] polyoxyethylene sorbitan monoleate solution soaking process: the mass fraction that the ratio of getting 2ml in every square centimeter of semi-finished product C gets respective amount is the polyoxyethylene sorbitan monoleate solution of 0.2%, semi-finished product C and polyoxyethylene sorbitan monoleate solution of getting are placed in same container, be positioned over shaking table and shake 30 minutes, change polyoxyethylene sorbitan monoleate solution, be again placed in shaking table and shake 30 minutes.Repeat above step, until when the pH value of the polyoxyethylene sorbitan monoleate solution displaced is between 6.0 ~ 7.0, discard polyoxyethylene sorbitan monoleate solution, obtain semi-finished product D;
Step [5] purified water immersion treatment: the ratio of getting 2ml in every square centimeter of semi-finished product D gets the purified water of respective amount, semi-finished product D and purified water of getting are placed in same container, be positioned over shaking table and shake 30 minutes, change purified water, be again placed in shaking table and shake 30 minutes.Repeat above step, until when the pH value of the purified water displaced is between 5.5 ~ 6.5, discard purified water, obtain semi-finished product E;
Step [6] Freamine Ⅲ immersion treatment: the ratio of getting 2ml in every square centimeter of semi-finished product E gets the aminoacid mixed solution mixed by glutamic acid and glycine of respective amount, containing glutamic acid 0.75g, glycine 1.5g in every 1000 milliliters of these aminoacid mixed solutions, semi-finished product E and aminoacid mixed solution of getting are placed in same container, put shaking table and shake 30 minutes, then 30 minutes are left standstill, discard aminoacid mixed solution, obtain semi-finished product F;
Step [7] purified water immersion treatment: the ratio of getting 2ml in every square centimeter of semi-finished product F gets the purified water of respective amount, semi-finished product F and purified water of getting are placed in same container, be positioned over shaking table and shake 30 minutes, change purified water, be again placed in shaking table and shake 30 minutes.Repeat above step, until when the pH value of the purified water displaced becomes between 6.0 ~ 7.0, discard purified water, obtain semi-finished product G;
Step [8] PBS buffer immersion treatment: the ratio of getting 2ml in every square centimeter of semi-finished product G gets the PBS buffer that respective amount pH value is 7.5, semi-finished product G and PBS buffer of getting are placed in same container, be positioned over shaking table and shake 30 minutes, then 30 minutes are left standstill, discard used PBS buffer, obtain semi-finished product H;
Step [9] is repaired and finishing: by clean for all foreign bodies removals in the corium face of semi-finished product H, epidermis side, the hole that diameter is more than 1 centimetre if exist, then with medical suture, hole is sewed on, during slot apertures hole, the distance controlled between stitch is not more than 1 centimetre, if shape irregularity, by its trimmed, obtain semi-finished product I;
Step [10] sodium chloride solution immersion treatment: the mass fraction that the ratio of getting 1ml in every square centimeter of semi-finished product I gets respective amount is the sodium chloride solution of 0.9%, then semi-finished product I and sodium chloride solution of getting are placed in same container, be positioned over shaking table and shake 30 minutes, change sodium chloride solution, be again placed in shaking table and shake 30 minutes.Repeat above step, until when the pH value of the sodium chloride solution displaced is between 6.5 ~ 7.5, take out semi-finished product I; The mass fraction that the ratio of getting 1ml in every 1 square centimeter of semi-finished product I gets respective amount is the sodium chloride solution of 0.9%, and semi-finished product I and sodium chloride solution of getting are placed in same container, leaves standstill immersion 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, seal packing inside bag, and then loading outer packaging bag and sealed, is the cobalt Co of 25kGy finally by irradiation dose
60irradiation, final finished product K.
One, as follows through the checking of glutaraldehyde solution immersion treatment technique deactivation HIV effect to allograft skin raw material in step described in above-described embodiment [1]:
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 (using front deionized water to be configured to 0.5%);
1.3 human immunodeficiency viruses: HIV-1 IIIB strain, are introduced by the U.S.;
1.4 MT
2cell: human T lymphocyte of going down to posterity is;
1.5 cell culture fluids: RPMI 1640+10% hyclone;
1.6 96 porocyte culture plates
1.7 CO
2incubator: MCO-15A.
2 methods
Get each one piece of (the about 1cm of raw material skin respectively
2/ block), PBS cyclic washing, abandons PBS, then adds HIV-1 IIIB 0.1ml, repeatedly blows and beats several times, hatches 30min, adds RPMI 1640 cell culture fluid 0.9ml, mixing, move in filtering centrifuge tube, 3000 revs/min 2 minutes, filtrate as zero time sampling; Get each one piece of (the about 1cm of raw material skin more respectively
2/ block), PBS cyclic washing, abandon PBS, then add HIV-1 IIIB 0.1ml, repeatedly blow and beat several times, hatch 30min, add 0.5% glutaraldehyde 0.9ml again, immersion treatment 50 minutes, moved in filtering centrifuge tube respectively at 10,20,30,50 minutes, 3000 revs/min 2 minutes, respectively filtrate is sampled; Every sub-sampling 0.1ml, the gradient dilution of 10 is made at once of culture fluid, measure the titre of wherein HIV, establish virus control, dilution contrast, positive control and cell controls simultaneously, the titre of HIV is measured with MT2 cell micro culture method, using cytopathy as differentiating the index that virus exists, carry out three duplicate detection to 1 batch sample, 2 and 3 batch samples respectively carry out one-time detection.
3 conclusions
Allograft skin raw material described in step of the present invention [1] adds HIV-1 IIIB, hatch 30 minutes, add 0.5% glutaraldehyde immersion treatment again 50 minutes, the titre of HIV-1 IIIB can be made to decline 4.17 log(testing results as shown in Figure 2) and through cell culture blind passage three generations, there is not cytopathy.
Two, as follows to production technology Validation of Virus Inactivation in Human of the present invention:
Checking foundation: No. [2002] 160, traditional Chinese medicines prison note, " animal derived medical device product registration declaration material writes guideline ", " allogeneic medical apparatus and instruments virus inactivation technology verification guide principle ".
Checking object: manufacturing process per sample, verify respectively 0.5% glutaraldehyde soak 50 minutes, 2 kinds of techniques such as 60Co-γ rays irradiation 25KGy are to the inactivating efficacy of selected 3 kinds of indicator viruses.
1. verification sample: continuous three batch samples taking from inactivation technology back in production process, in 4 DEG C of preservations.
2. indicator virus is selected:
(1) Pseudorabies virus (PRV): belong to herpetoviridae, for double-stranded DNA has togavirus.To the fatsolvent such as ether, chloroform, the sensitivity such as formalin and ultraviolet radiation is the one that in herpesvirus, resistance is stronger.For the HBV indicator virus of traditional Chinese medicines prison note [2002] No. 160 literary composition regulations.
(2) Xin get Bi Shi virus (Sindbis): belong to Alphaherpesvirinae, ball-type, for RNA has togavirus.Lower to the resistance of physical and chemical factor.For the HCV indicator virus of traditional Chinese medicines prison note [2002] No. 160 literary composition regulations.
(3) pig parvoviral (PPV): belong to Parvoviridae, for small DNA is without togavirus, 18-24nm.Due to without cyst membrane, very strong to physical and chemical factor resistance.For the non-fat peplos assays for parvovirus B 19 indicator virus of traditional Chinese medicines prison note [2002] No. 160 literary composition regulations.
3. inactivation of virus/removal verification step:
(1) 0.5% glutaraldehyde solution soaks 50 minutes inactivation technologies:
Allogenic material sample is put into the culture bottle after 50ml sterilization, adds 0.5% glutaraldehyde solution 18ml respectively, 2ml indicator virus solution (10%).Respectively at sampling in 0,5,10,20,30,50 minute, result maintenance medium according to cell toxicity test does the residual titre of suitably dilution rear detection virus, both can stop the continuation deactivation of 0.5% glutaraldehyde solution, 0.5% glutaraldehyde solution can have been eliminated again to the toxicity detecting cell.Often kind of sample does three batch weight again.
(2) 60Co-γ rays irradiation 25KGy inactivation technology:
Allogenic material sample is put into the culture bottle after 50ml sterilization, adds 20ml indicator virus solution respectively and directly soak.Take out 1ml immediately and do suitably dilution for predose correspondence, all the other samples are 60Co-γ rays irradiation 25KGy.Irradiation terminates rear sample thief and measures the residual titre of virus with the same dilution factor of predose sample.Often kind of sample does three batch weight again.
4. virus activity titration method:
(1) cytotoxicity detects:
In order to the initial dilution after determining 0.5% glutaraldehyde solution deactivation during the residual titre of sample determination virus, first must do the different dilution factor of 0.5% glutaraldehyde solution to the toxicity test detecting cell, just can carry out virus activity titration.But by diluting the continuation deactivation of termination 0.5% glutaraldehyde solution.Cytotoxicity detects and uses MTT colorimetry.
(2) virus activity titration:
Select Vero and the PK-15 cell strain that virus is responsive, adopt Microdose cytopathic effect assay.The sample got by different time in above-mentioned each group, according to cytotoxicity testing result, determines the initial dilution of sample, then after doing ten times of serial dilutions, add in 96 orifice plates inoculated and detected cell immediately, each dilution factor does 8 holes and repeats, and puts 37 DEG C, hatches in 5%CO2 incubator.With Vero cell detection PRV and Sindbis virus incubation 72 hours, light Microscopic observation also records cytopathy (CPE) situation.Because the cytopathogenic effect of PPV is more weak, so on the basis that need judge in cytopathy, increase specificity anti-PPV fluorescent antibody staining and at fluorescence microscopy Microscopic observation.The emerald green fluorescence speckle that the cell infected by PPV is beautiful as seen when there is not pathological changes, the method can significantly improve the specificity and sensitivity that PPV detects.Virus titer is pressed KarberShi method and is calculated.
5. result judges:
Virus reducing amount >=4LgTCID50/ml, judges that this viral inaction steps is effective.
6. cell blind passage three generations:
Often criticizing the equal high concentration of sample after various virus inactivation technology deactivation adds in passage cell, does cell blind passage 3 generation, observes whether occur cytopathy.In the overall process in cell blind passage 3 generation, any period occurs that cytopathy is the positive (+), virus is described not by complete inactivation; Do not occur that cytopathy is negative (-), virus is described 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 that two kinds are detected cell
Table 1 0.5% glutaraldehyde solution is on the impact detecting Growth of Cells
8. 0.5% glutaraldehyde solution process different time is to the observation of three kinds of model virus inactivating efficacies in " allogenic material sample ":
8.1 0.5% glutaraldehyde solution process different times are to the inactivating efficacy of PRV virus: (as shown in table 2, table 3)
Table 2 0.5% glutaraldehyde solution process different time is to the inactivating efficacy of PRV virus
Table 3 0.5% glutaraldehyde solution soaks blind passage result after 50 minutes deactivation PRV virus
| Sample number | A blind passage generation | Blind passage is secondary | Blind passage three generations |
| 1 | （-） | （-） | （-） |
| 2 | （-） | （-） | （-） |
| 3 | （-） | （-） | （-） |
| Cell controls | （-） | （-） | （-） |
8.2 0.5% glutaraldehyde solutions soak different time to the inactivating efficacy of Sindbis virus: (as shown in table 4, table 5)
Table 4 0.5% glutaraldehyde solution soaks different time to the inactivating efficacy of Sindbis virus
Table 5 0.5% glutaraldehyde solution soaks blind passage result after 50 minutes deactivation Sindbis virus
| Sample number | A blind passage generation | Blind passage is secondary | Blind passage three generations |
| 1 | （-） | （-） | （-） |
| 2 | （-） | （-） | （-） |
| 3 | （-） | （-） | （-） |
| Cell controls | （-） | （-） | （-） |
8.3 0.5% glutaraldehyde solutions soak different time to the inactivating efficacy of PPV virus: (as shown in table 6, table 7)
Table 6 0.5% glutaraldehyde solution soaks different time to the inactivating efficacy of PPV virus
Table 7 0.5% glutaraldehyde solution soaks blind passage result after 50 minutes deactivation PPV virus
| Sample number | A blind passage generation | Blind passage is secondary | Blind passage three generations |
| 1 | （-） | （-） | （-） |
| 2 | （-） | （-） | （-） |
| 3 | （-） | （-） | （-） |
| Cell controls | （-） | （-） | （-） |
9. 60Co-γ rays irradiation 25KGy process " allogenic material sample " observation to three kinds of model virus inactivating efficacies:
9.1 60Co-γ rays irradiation 25KGy process " allogenic material sample " are to PRV inactivation of virus effect: (as shown in table 8,9)
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 deactivation PRV
| Sample number | A blind passage generation | Blind passage is secondary | 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 processing sample material is to Sindbis inactivation of virus effect
Blind passage result after table 11 60Co-γ rays irradiation 25KGy processing sample material deactivation Sindbis
| Sample number | A blind passage generation | Blind passage is secondary | Blind passage three generations |
| 1 | （-） | （-） | （-） |
| 2 | （-） | （-） | （-） |
| 3 | （-） | （-） | （-） |
| Cell controls | （-） | （-） | （-） |
9.3 60Co-γ rays irradiation 25KGy process " allogenic material sample " are to PPV inactivation of virus effect: (as shown in table 12,13)
Table 12 60Co-γ rays irradiation 25KGy processing sample material is to PPV inactivation of virus effect
Blind passage result after table 13 60Co-γ rays irradiation 25KGy processing sample material deactivation PPV
| Sample number | A blind passage generation | Blind passage is secondary | Blind passage three generations |
| 1 | （-） | （-） | （-） |
| 2 | （-） | （-） | （-） |
| 3 | （-） | （-） | （-） |
| Cell controls | （-） | （-） | （-） |
10. conclusion:
Demonstration test result shows, and two virus inactivation technologies in the production of product prepared by the present invention all can effective three kinds of model virus adding of deactivation.0.5% glutaraldehyde solution soaks 50 minutes technique, and processing 5 minutes can complete inactivation PRV, Sindbis virus.Because PPV is without peplos parvovirus, substantially not containing albumen, extremely strong to chemical factors patience, need 30 minutes this virus of process ability complete inactivation.Virus reducing amount is respectively: PRV virus>=6.0-6.13 LgTCID
50; Sindbis virus>=5.13-5.5 LgTCID
50; PPV virus>=4.75-4.88 LgTCID
50.0.5% glutaraldehyde solution soaks the adding cell blind passage three generations flaviviridae viral detect containing virus sample of process in 50 minutes.60Co-γ rays 25KGy irradiation technique can deactivation PRV virus>=6.38-7.13 LgTCID
50; Sindbis virus>=5.75-5.88 LgTCID
50; PPV virus>=6.0-6.25LgTCID
50.Three kinds of virus sample of irradiation 25KGy detect through cell blind passage three generations flaviviridae viral.
According to the regulation of No. [2008] 7, state's food medicine prison note with traditional Chinese medicines prison note [2002] No. 160 files, often walking in inactivation technology, viral reducing amount reaches more than 4logs, can think that inactivation of viruses method is effective.And each technique can superpose the deactivation of virus.Virus reducing amount is larger, and blind passage three generations is virus-free detects, and illustrate that inactivation technology effect is better, the safety of product is higher.This result of the test shows, two techniques of checking, and viral reducing amount is all greater than 4logs, if two kinds of technique superpositions, the reducing amount of three kinds of indicator viruses all reaches more than 10logs.
So, 0.5% glutaraldehyde solution in preparation method of the present invention soak 50 minutes, 60Co-γ rays 25KGy irradiation two-strain inactivation technology can ensure the Viral safety of this series products.
Three, as follows to the immunogenicity checking of the human-body biological dressing made by embodiment of the present invention:
Checking foundation: animal derived medical device product registration declaration material writes guideline
Checking object: by implanting 2 weeks, 4 weeks, 12 weeks serum antibody IgG, IgA, IgM situations of change after dressing of the present invention to rabbit, the immunogenicity of product prepared by evaluation the present invention.
1. laboratory sample: the biological human biological dressing that the manufacture method provided according to the embodiment of the present invention is produced, product batch number: 1 and 2, is placed in buffer 4 DEG C of refrigerators and deposits.
2. laboratory animal, reagent and instrument and equipment
Laboratory animal: white big ear rabbit 60, body weight 2.05 ± 0.21kg, hero is female not to be limit.
Main agents: rabbit igg, IgA, IgM ELISA kit, T96, T48 two kinds of specifications; Immunological adjuvant, 10ml/ props up; 5% pentobarbital injection; Gentamicin injection liquid, 80,000 IU/2ml.
Key instrument equipment: agitator; Centrifuge; Refrigerator; Electro-heating standing-temperature cultivator; Microplate reader.
3. blood specimen collection
Experiment is front and test rear 2 weeks, 4 weeks, 12 weeks blood sample collections.Gather non-anticoagulation 1ml with asepsis injector through rabbit arteria auricularis, be placed in about 1h under room temperature, after solidifying, put 4 DEG C of refrigerator overnight and separate out serum, centrifugal 10min under 3500rpm, sucking-off serum, is sub-packed in EP pipe, is stored in-80 DEG C of refrigerators after labelling cleaning.After all having taken a blood sample after 12 weeks, IgG antibody, IgA, IgM horizontal detection are carried out in unification.
4. the preparation of biological human biological dressing leachate
Shredded by human-body biological dressing in super-clean bench, be placed in blender, add the phosphate buffer of sterilized Ph7.0, stir into homogenate, in experiment, apparatus used is all through autoclaving process.Homogenate is placed in after 37 DEG C of electro-heating standing-temperature cultivators place and spend the night, and centrifugal 10min under 12000rpm, gets supernatant degerming through the sterile filter of 0.22 μm.Put in 4 DEG C of refrigerators and deposit.The preparation in 1 day before use of human-body biological dressing leachate.
5. experiment grouping and process
Experimental rabbit (60) is divided into four groups: high dose group (implant into body biological dressing group), low dose group (leachate group), negative control group, positive controls (booster immunization group).Often organize 15.
High dose group (implant into body biological dressing group): rabbit 5% anaesthetized with pentobarbital, dosage is 0.5ml/kg, slowly injects through ear edge tremulous pulse.After the depilation of rabbit back local skin, sterilization, at incision of skin, human-body biological dressing is implanted in skin, sew up process.
Low dose group (leachate group): get human-body biological dressing leachate 0.7ml, points 6 to be injected in rabbit vertebra both sides subcutaneous, injects about 0.12ml at often.
Negative control group: modus operandi is identical with high dose group, but not implant into body biological dressing.
Positive controls (booster immunization group):
The preparation of human-body biological dressing leachate and adjuvant mixed liquor: human-body biological dressing leachate is mixed with adjuvant equal-volume, mixed liquor is sucked respectively in the sterilizing glass syringe of two 100ml, the two syringes aseptic plastic pipe that is about 10cm connects, push-and-pull two syringe makes liquid blending respectively, forms water in oil emulsion.Emulsion being dripped one drops on the water surface, Emulsion keep tear drop complete and do not disperse to be qualified.Human-body biological dressing leachate and adjuvant mixed liquor are prepared before use.
Human-body biological dressing leachate immunizing rabbit with adding adjuvant: get human-body biological dressing leachate and adjuvant mixed liquor 1.5ml, points 6 to be injected in rabbit vertebra both sides subcutaneous, injects about 0.25ml at often.After 2 weeks, then use the human-body biological dressing leachate of same dose and adjuvant mixed liquor booster immunization once, method is the same.
For preventing postoperative infection, carry out the rabbit (high dose group and negative control group) of performing the operation at the operation same day and postoperative 3 days intramuscular injection gentamicin injection liquid, 40,000 units/sky.
Gather the experiment blood sample of latter 2 weeks, 4 weeks, 12 weeks.In experimentation, observe animal wound healing situation, ooze out situation with or without redness; Situation and the animals survived situations such as animal activity, feed, drinking-water.
6. ELISA detects
Serum antibody IgG, IgA, IgM horizontal detection operates according to test kit description.Assay method is as follows:
(1) dilution of standard substance and application of sample: standard substance are diluted according to the Serum antibody concentrations scope that preliminary experiment obtains.The dilution of IgG standard substance is 1.5,3,6,12,18 μ g/ml, five concentration point, and it is 0.75,1.5,3,6,9 μ g/ml, five concentration point that IgA, IgM standard substance all dilute.50 μ l standard dilutions are added enzyme mark bag by plate gauge orifice.
(2) application of sample: establish blank well (blank control wells does not add sample and enzyme marking reagent, and respectively step operation is identical for all the other), testing sample hole respectively.In enzyme mark bag is by testing sample hole on plate, first add sample diluting liquid 40 μ l, and then to add the final dilution factor of testing sample 10 μ l(sample be 5 times).
(3) incubation: with the rearmounted 37 DEG C of incubation 30min of shrouding film shrouding.
(4) cleaning mixture is prepared: by for subsequent use after 30 times of concentrated cleaning solution distilled water 30 times dilution.
(5) wash: carefully take shrouding film off, discard liquid, dry, cleaning mixture is filled it up with in every hole, discards, repeat 5 times, pat dry after leaving standstill 30s.
(6) enzyme-added: every hole adds enzyme marking reagent 50 μ l, except blank well.
(7) incubation: operation is with (3).
(8) wash: operation is with (5).
(9) develop the color: every hole first adds developer A50 μ l and adds developer B50 μ l again, shakes mixing gently, 37 DEG C of lucifuge colour developing 15min.
(10) stop: every hole adds stop buffer 50 μ l, cessation reaction.
(11) measure: with blank well zeroing, 450nm wavelength sequentially measures the absorbance in each hole.Mensuration should be carried out within 15min after adding stop buffer.
Three wells detects, and result of averaging judges.
7. statistical analysis
SPSS16.0 software is adopted to carry out statistical analysis.Measurement data is used
represent, between each group and the contrast of the antibody test result of different sampling stages adopt one factor analysis of variance inspection, the comparison of rate adopts χ
2detect.All statistical inferences all adopt two-sided test, and the inspection level with statistical significance is decided to be P<0.05.
8. immunogenicity experiments result
The ordinary circumstance of 8.1 laboratory animals
Carry out the rabbit visible movable minimizing in a day after surgery of performing the operation, feed reduces, and recovers normally, to occur without wound infection later, and wound is without red and swollen transudate, and animal wound healing is good, and obvious abnormal response does not appear in No operation rabbit.Each group of dead animal number is in table 14.
Table 14 is group dead animal number respectively
χ is carried out to each treated animal mortality rate
2detect, obtain χ
2=0.373, P=0.946>0.05, shows rabbit mortality rate no difference of science of statistics between each group, and the death of rabbit and human-body biological dressing are implanted irrelevant.
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 lists in table 15.
Table 15 Serum Antibodies of Rabbits IgG, IgA, IgM level (μ g/ml)
8.2.2 the change of the rear different time Serum Antibodies of Rabbits of process
After process, Fig. 3 ~ 5 are shown in the change of different time Serum Antibodies of Rabbits.As can be seen from the figure, each group Serum Antibodies of Rabbits level all raises at second week, decline to some extent, but still higher than the foundation level before experiment, the 12nd week serum antibody is reduced to the foundation level before experiment substantially in 4th week.
Carry out statistical test to different time points antibody level of serum, result lists table 16-19 in.As can be seen from results of statistical analysis, high dose group and positive controls after treatment second week IgG, IgA, IgM level, all higher than level before experiment, have the meaning of significance,statistical; After low dose group process, second week IgA and IgM level are higher than the level before experiment, and the level before IgG and experiment contrasts no difference of science of statistics; Negative control group after treatment second week IgG level, also higher than the level before experiment, may cause slight immunoreation relevant with operation wound; High dose group and positive controls after treatment 4th week IgA and IgM level are still significantly higher than the level before experiment; The each group of level all substantially returned to for the 12nd week after treatment before experiment, with the 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 the contrast of Serum Antibodies of Rabbits level is respectively organized
The 2nd week difference is more obvious after treatment for each group of Serum Antibodies of Rabbits level (Fig. 6-9), within 2nd week, respectively organize Serum Antibodies of Rabbits level and see Fig. 7, as can be seen from the figure, 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 of Serum Antibodies of Rabbits level, the results are shown in table 20-22.As can be seen from the table, after process, the 2nd week high dose group IgG, IgA, IgM level are all higher than negative control group, within 4th week, IgA and IgM level is still higher than negative control group, result has statistical significance, illustrate that human-body biological dressing is implanted and can cause rabbit immunoreation to a certain degree, but antibody is still in reduced levels, IgG, IgA, IgM top level is only respectively 25.93,10.16,9.98 μ g/ml.Adopt adjuvant booster immunization, contrast with high dose group, have no antibody horizontal and significantly improve, show that human-body biological dressing is after going antigen process, immunogenicity is very faint, even if adopt adjuvant booster immunization, also can not cause obvious immunoreation.
The results of statistical analysis of rabbit anteserum IgG level respectively organized by table 20
The results of statistical analysis of rabbit anteserum IgA level respectively organized by table 21
The results of statistical analysis of rabbit anteserum IgM level respectively organized by table 22
9. conclusion
After 9.1 implant into body biological dressings there is not obvious abnormal response in rabbit, and it is safe for showing that human-body biological dressing is implanted;
9.2 serum antibodys reach top level in 2 weeks after human-body biological dressing is implanted, and decrease after 4 weeks, close to the foundation level before experiment when 12 weeks;
9.3 human-body biological dressing implant rear 2nd week antibody level of serum higher than negative control group, within 4th week, IgA and IgM level is still higher than negative control group, illustrate that human-body biological dressing can cause rabbit immunoreation to a certain degree for 2 ~ 4 weeks after implanting, but antibody is still in lower expression, IgG, IgA, IgM top level is only respectively 25.93,10.16,9.98 μ g/ml; Adopt adjuvant booster immunization, also have no antibody horizontal and significantly improve, show that the immunogenicity of human-body biological dressing is very weak.
Four, to the human-body biological casting product clinical trial one made by embodiment of the present invention:
Clinical physical data (selection of sick kind, total cases and case): this group burns (wound) sick and wounded people 30 example, wherein man 21 example, and female 9 is routine; Age 2-58 year; Minimum area 1%, maximum area 5%; Totally 36 wound surface, wherein fresh shallow II ° of wound surface 12, fresh dark II ° wound surface 6, residual wound 6, cuts (cutting) crust skin grafted wounds 6,6, skin donor site.This group case is the outpatient service same period and inpatient.
Clinical testing procedure: this group experimental technique is undertaken by clinical trial protocol, test group uses the human-body biological dressing prepared by the present invention to cover all kinds of burning (wound) wound surface, and matched group selects Petroleum gauze or other biological dressing.Test group and matched group select the same position of same patient. or the wound surface of different parts carries out comparitive study.
The evaluation criterion taked is or/and statistical method: the conventional healing time of wound surface compares with test group healing time. select the process of T test statistics method, as the foundation of the human-body biological dressing curative effect evaluated prepared by the present invention.Covering is cut (cutting) the autologous little skin graft of crust or wear debris emission wound surface, postoperative more than 5 weeks use human body biological dressing gradually drying come off, the wound surface person of closing is considered as excellent; Within less than 4 weeks, person is considered as poor bad.
Clinical test results: this group 30 example, 36 wound surface, only 1 children's's wound surface is changed loosely and again because of fixing.The healing in general 8-10 days of fresh shallow II ° of wound surface, shorten in average 2-4 days; Healing in the general 13-17 of dark II ° wound surface days, shorten in average 3-5 days; Wound surface NIP reacts.Granulation wound, chronic ulcer, decubital ulcer use, slough, purification, clean wound surface can be removed, be beneficial to green meat blastogenesis long, wound healing or cultivated good wound base for skin-grafting reparation.Boundary lid is cut (cutting) crust Micrograiting wound surface, human-body biological dressing mummification used in postoperative more than 5 weeks comes off, and improves healing quality.Wound surface is checked, without clear scar hypertrophy phenomenon after 2 months; And do not use the human-body biological dressing person prepared by the present invention, there is scar hyperplasia phenomenon.Laboratory examination: blood, routine urinalysis and Liver and kidney function are without exception, thin rice seedling is cultivated without growth; Clinical practice has no toxic and side effects.
Clinical trial result is analyzed: the human-body biological dressing prepared by the present invention protects wound surface effectively, asks when shortening healing: for epidermis avulsion wound surface, can form " seamless " and immediately cover; Especially the technical finesse of removing antigen has been carried out in dermal tissue face, reduce antigen dose, cut (cutting) crust microgranule transplant plane and upper use, strong adhesion, histocompatibility are good, extend the time of flap coverage, improve the outward appearance after wound healing and function.
Clinical trial conclusion: the human-body biological casting product prepared by the present invention, is applicable to cover all kinds of burning (wound) and hinders wound surface and Micrograiting wound surface, effectively protect wound surface, blocks fourth antibacterial and invades eight, shorten healing time; The patient that skin donor site extremely lacks, first covers human-body biological dressing of the present invention and can provide the guarantee of time for skin-grafting next time.There is the effect of purification, clean ulcer, decubital ulcer and granulation wound, promote fresh granulation tissue growth, wound surface can spontaneous recovery or for skin-grafting repair cultivated good epithelial cell growth environment, be a kind of wound surface external human-body biological dressing of the novel concept be worthy to be popularized.
Five, to the human-body biological casting product clinical trial two made by embodiment of the present invention:
Clinical physical data (selection of sick kind, total cases and case): this group all selects outpatient service and inpatient 32 example of same time period: man 18 example, female 14 example; Age 1-36 year; Totally 42 wound surface: shallow II ° example (8 wound surface), dark II ° example (8 wound surface), large area III ° of 5 example (15 wound surface); Granulation wound 4 example (4 wound surface), chronic ulcer 2 example (2 wound surface), decubital ulcer 2 example (2 wound surface); Fresh wound and wound surface 3 example (3 wound surface).This group uses minimum area 6cm × 4 cm, maximum area 50cm × 40cm.
Clinical testing procedure (comprise matched group arrange): 1, test group and matched group case adopt random packet: test group uses the dressing of the embodiment of the present invention made human-body biological to cover all kinds of burning (wound) to hinder wound surface, and the wound surface that matched group cladding material selects Petroleum gauze or biological dressing 2, selection consubstantiality or same site tissue damage to be similar to comparing as test group and matched group curative effect.
The evaluation criterion taked is or/and statistical method: 1, with the conventional healing time of all kinds of wound surface for standard, with test group heal platform time compared with, the statistical method process of selecting T to check, as the foundation evaluating human-body biological dressing of the present invention shortening wound healing time; 2, cover Micrograiting wound surface, within postoperative more than 5 weeks, come off, it is up to standard qualified to be considered as; Within less than 4 weeks, come off for defective.
Clinical test results: this group 32 example, 42 wound surface, wherein 2 wound surface are changed loosely and again because of fixing.Shallow II ° of wound surface 7-9 days is healed platform, shortens 3-5 days; Dark II ° wound surface about 2 weeks healing, shortens 5-7 days; Have no inflammatory reaction.Granulation wound, chronic ulcer, decubital ulcer use, slough, purification, clean wound surface can be removed, be beneficial to green meat blastogenesis long, wound healing or cultivated good wound base for making skin graft.Blood, routine urinalysis and Liver and kidney function no abnormality seen, antibacterial culturing, without growth, has no toxic and side effects.Cover III ° and cut (cutting) crust Micrograiting wound surface, within postoperative more than 5 weeks, come off, improve healing quality.
Clinical trial result is analyzed: the human-body biological dressing prepared by the present invention has the advantage of allograft skin flap coverage, for wound repair creates good internal environment, shortens wound healing time.In addition, the dermal tissue face of the human-body biological dressing prepared by the present invention, through the technical finesse of past antigen, has the effect of skim extracellular matrix " support ", improves outward appearance and the function of the rear wound surface of healing.
Clinical trial conclusion: the human-body biological dressing prepared by the present invention, be applied to all kinds of burning of covering (wound) and hinder wound surface and Micrograiting wound surface, there is good covering effect, alleviate wound pain, promote epithelial growth, shorten healing time, reduce cicatrization; In addition, cover ulcer, decubital ulcer and granulation wound and, can purify, clean wound surface, promote that green meat blastogenesis is long, be beneficial to wound healing or cultivated good wound base for skin-grafting reparation.A kind of wound surface external human-body biological dressing of the novel concept be worthy to be popularized.
Above-describedly be only the preferred embodiments of the present invention; be understood that; the explanation of above embodiment just understands method of the present invention and core concept thereof for helping; the protection domain be not intended to limit the present invention; all any amendments, equivalent replacement etc. made within thought of the present invention and principle, all should be included within protection scope of the present invention.
Claims (11)
1., for covering a manufacture method for the human-body biological dressing of burn trauma wound surface, comprise the following step carried out in order:
The mass fraction that step [1] gets respective amount in the ratio that every square centimeter of allograft skin gets 0.5 ~ 3ml is the glutaraldehyde solution of 0.25% ~ 3%, allograft skin is put into got glutaraldehyde solution, soaks 15 ~ 60 minutes; Change glutaraldehyde solution, continue immersion 15 ~ 30 minutes; Take out allograft skin, the mass fraction that the ratio of getting 0.5 ~ 3ml in every square centimeter of allograft skin gets respective amount is the sodium chloride solution of 0.5% ~ 1.5%, allograft skin is put into got sodium chloride solution, soaks 30 ~ 120 minutes, and taking-up allograft skin obtains semi-finished product A;
Semi-finished product A is split by required specification by step [2], carries out anti-take, remove remaining fatty tissue, obtain semi-finished product B with bark fetching drum to the semi-finished product A after segmentation;
Step [3] gets the purified water of respective amount in the ratio that every square centimeter of semi-finished product B gets 0.5 ~ 3ml, semi-finished product B and purified water of getting are placed in same container, be positioned over shaking table concussion 15 ~ 60 minutes, change purified water, be again placed in shaking table concussion 15 ~ 60 minutes; Repeat above step, until when the pH value of the purified water displaced is between 5.5 ~ 6.5, discard purified water, obtain semi-finished product C;
Step [4] is the polyoxyethylene sorbitan monoleate solution of 0.05% ~ 0.3% in the mass fraction that the ratio that every square centimeter of semi-finished product C gets 0.5 ~ 3ml gets respective amount, semi-finished product C and polyoxyethylene sorbitan monoleate solution of getting are placed in same container, be positioned over shaking table concussion 15 ~ 60 minutes, change polyoxyethylene sorbitan monoleate solution, be again placed in shaking table concussion 15 ~ 60 minutes; Repeat above step, until when the pH value of the polyoxyethylene sorbitan monoleate solution displaced is between 6.0 ~ 7.0, discard polyoxyethylene sorbitan monoleate solution, obtain semi-finished product D;
Step [5] gets the purified water of respective amount in the ratio that every square centimeter of semi-finished product D gets 0.5 ~ 3ml, semi-finished product D and purified water of getting are placed in same container, be positioned over shaking table concussion 15 ~ 60 minutes, change purified water, be again placed in shaking table concussion 15 ~ 60 minutes; Repeat above step, until when the pH value of the purified water displaced is between 5.5 ~ 6.5, discard purified water, obtain semi-finished product E;
Step [6] gets the Freamine Ⅲ of respective amount in the ratio that every square centimeter of semi-finished product E gets 0.5 ~ 3ml, semi-finished product E and Freamine Ⅲ of getting are placed in same container, put shaking table concussion 15 ~ 60 minutes, then leave standstill 15 ~ 30 minutes, discard Freamine Ⅲ, obtain semi-finished product F;
Step [7] gets the purified water of respective amount in the ratio that every square centimeter of semi-finished product F gets 0.5 ~ 3ml, semi-finished product F and purified water of getting are placed in same container, be positioned over shaking table concussion 15 ~ 60 minutes, change purified water, be again placed in shaking table concussion 15 ~ 60 minutes; Repeat above step, until when the pH value of the purified water displaced becomes between 6.0 ~ 7.0, discard purified water, obtain semi-finished product G;
Step [8] gets the PBS buffer of respective amount in the ratio that every square centimeter of semi-finished product G gets 0.5 ~ 3ml, semi-finished product G and PBS buffer of getting are placed in same container, be positioned over shaking table concussion 15 ~ 60 minutes, then 15 ~ 30 minutes are left standstill, discard used PBS buffer, obtain semi-finished product H;
Step [9] is clean by all foreign bodies removals in the corium face of semi-finished product H, epidermis side, and the hole that diameter is more than 1 centimetre if exist, then sew on hole with medical suture, if shape irregularity, by its trimmed, obtains semi-finished product I;
The mass fraction that step [10] gets respective amount in the ratio that every square centimeter of semi-finished product I gets 0.5 ~ 3ml is the sodium chloride solution of 0.5% ~ 1.5%, then semi-finished product I and sodium chloride solution of getting are placed in same container, be positioned over shaking table concussion 15 ~ 60 minutes, change sodium chloride solution, be again placed in shaking table concussion 15 ~ 60 minutes; Repeat above step, until when the pH value of the sodium chloride solution displaced is between 6.5 ~ 7.5, take out semi-finished product I; The mass fraction that the ratio of getting 1ml in every 1 ~ 3 square centimeter of semi-finished product I gets respective amount is the sodium chloride solution of 0.5% ~ 1.5%, and semi-finished product I and sodium chloride solution of getting are placed in same container, leaves standstill immersion 15 ~ 60 minutes, discards solution, obtain semi-finished product J;
Semi-finished product J is loaded packing inside bag by step [11], paves in packing inside bag, is sealed by packing inside bag, then loads outer packaging bag and is sealed, finally by cobalt Co
60irradiation, final finished product K.
2. the manufacture method of a kind of biological dressing for covering burn trauma wound surface according to claim 1, it is characterized in that: allograft skin described in step [1] derives from the skin that healthy human body contributes body, its thickness is 0.2 ~ 0.3 millimeter, the thickness difference of getting between allograft skin be no more than 0.1 millimeter.
3. the manufacture method of a kind of biological dressing for covering burn trauma wound surface according to claim 1 and 2, it is characterized in that: in step [1] use the mass fraction of glutaraldehyde solution to be 0.5%, use glutaraldehyde solution to get 1ml in every square centimeter of allograft skin ratio obtain, the first time soak time of described allograft skin in glutaraldehyde solution is 50 minutes, and second time soak time is 25 minutes.
4. the manufacture method of a kind of biological dressing for covering burn trauma wound surface according to claim 3, it is characterized in that: in step [4] use mass fraction be 0.2% polyoxyethylene sorbitan monoleate solution, the ratio that the polyoxyethylene sorbitan monoleate solution used gets 2ml in every square centimeter of semi-finished product C obtains, and the time that semi-finished product C shakes in got polyoxyethylene sorbitan monoleate solution on shaking table is at every turn 30 minutes.
5. the manufacture method of a kind of biological dressing for covering burn trauma wound surface according to claim 1 or 4, it is characterized in that: the Freamine Ⅲ described in step [6], wherein contained aminoacid is one or several in the middle of glutamic acid, alanine, glycine, aspartic acid, in the middle of every 1000 milliliters of Freamine Ⅲs, the content of one or several of glutamic acid, alanine, glycine, aspartic acid is respectively 0.1 ~ 3g respectively.
6. the manufacture method of a kind of biological dressing for covering burn trauma wound surface according to claim 5, it is characterized in that: the aminoacid contained by Freamine Ⅲ described in step [6] is glutamic acid and glycine, in the middle of every 1000 milliliters of these Freamine Ⅲs, containing glutamic acid 0.75g, containing glycine 1.5g.
7. the manufacture method of a kind of biological dressing for covering burn trauma wound surface according to claim 1, is characterized in that: the pH value of the PBS buffer used in step [8] is 7.0 ~ 8.0.
8. the manufacture method of a kind of biological dressing for covering burn trauma wound surface according to claim 7, is characterized in that: the pH value of the PBS buffer described in step [8] is 7.5.
9. the manufacture method of a kind of biological dressing for covering burn trauma wound surface according to claim 1, is characterized in that: when meeting hole with medical suture described in step [9], the distance that should control between stitch is not more than 1 centimetre.
10. the manufacture method of a kind of biological dressing for covering burn trauma wound surface according to claim 1, is characterized in that: through cobalt Co in step [11]
60the dosage of irradiation is: 20kGy≤irradiation dose≤30kGy.
The manufacture method of 11. a kind of biological dressings for covering burn trauma wound surface according to claim 10, is characterized in that: through cobalt Co in step [11]
60the dosage of irradiation is: irradiation dose equals 25kGy.
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| CN1788801A (en) * | 2005-12-06 | 2006-06-21 | 天津市医药科学研究所 | Dismount free hurt covering with biocompatibility and its preparation method |
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| KR101089614B1 (en) * | 2010-02-26 | 2011-12-05 | (주)시지바이오 | Method for preparing acellular dermal matrix and acellular dermal matrix prepared therefrom |
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| CN1788801A (en) * | 2005-12-06 | 2006-06-21 | 天津市医药科学研究所 | Dismount free hurt covering with biocompatibility and its preparation method |
| CN101020077A (en) * | 2007-03-23 | 2007-08-22 | 四川大学 | Prepn process of collagen-based surface wound repairing membrane possessing tissue induction |
| CN102573945A (en) * | 2009-09-11 | 2012-07-11 | 翰林大学校产学协力团 | Production method for cryopreserved acellular dermal matrix, and cryopreserved acellular dermal matrix produced thereby |
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