CN1213774C - Technology for preparation of biological dressing material - Google Patents
Technology for preparation of biological dressing material Download PDFInfo
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- CN1213774C CN1213774C CNB991077741A CN99107774A CN1213774C CN 1213774 C CN1213774 C CN 1213774C CN B991077741 A CNB991077741 A CN B991077741A CN 99107774 A CN99107774 A CN 99107774A CN 1213774 C CN1213774 C CN 1213774C
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Abstract
The present invention relates to a technology for preparing a biological dressing, which has the steps: extracting bioactive substances and collagen which can promote the growth of epithelial cells from the epithelial and dermal tissue of a juvenile healthy mammal at low temperature by a biochemistry technology, and then combining the bioactive substances and the collagen with a traditional gauze dressing to obtain a biological dressing. The biological dressing has the characteristics of high effective efficiency, low cost and convenient use. Researches on animal experiments and clinical experiments prove that the dressing can obviously promote the healing of wounded surfaces of burn, chronic ulcer of skin, operation wound and various other skin injury diseases, enhance healing quality, shorten healing time and have no obvious adverse reaction or side effects.
Description
What the present invention relates to is a kind of biological dressing, and specifically, what the present invention relates to is the biological dressing that a kind of material that adopts the biologically active that extracts in mammal epithelium and the dermal tissue is made.
We manufacture experimently out a kind of novel surgical dressing by consulting document and clinical groping.The main material of this dressing is growing animal peel extract and hospital gauze, and it has drawn the advantage of collagen and gauze dressing.This product to the development of wound surface, lapse to and prognosis all has certain influence, it is mainly used in the multiple traumas such as burn, chronic ulcer and surgical wound surface, to reach the purpose that prevents wound infection, accelerating wound clinically.
The dressing of clinical practice at present mainly contains following several types:
(1), abiotic active dressing: i.e. traditional dressing.As the dressing of all kinds of gauze type, comprise synthetic fibers gauze, the thin property anti-adhesive gauze of plastics, wettability gauze etc.The major function of this class dressing is to prevent wound infection, the protection wound surface, but little to the effect that promotes wound healing, even also can influence wound healing.
(2), vegetable active dressing: the raw material of this class dressing is the organic component that extract from all kinds of plant varieties.As alginic acid salt, silicone, hydrocolloid class dressing (Duoderm dressing, Comfeel dressing), polysaccharide class dressings.The characteristics of this class dressing are that it can keep the wound moist environment, can act on mutually with the medicament and the body endogenous molecule of topical application, promote wound healing.There are some researches show: can keep moistening dressing can keep the hydras of wound surface.When the wound slough being carried out debridement with enzyme preparation, but because the activity of moistening environment kinase, thereby the effect of promotion enzyme.In addition this class dressing also with wound on molecule work, strengthen the effect of the self-sow factor in the wound repair, thus accelerating wound healing.
(3), the raw material of this class dressing of biotype biological dressing is the biotic component that extract from all kinds of biological varieties, is to take from viable tissue in the animal body.Can longerly preserve after treatment, become inanimate biomaterial.As amniotic membrane, peritoneum and skin.This class dressing has stronger antigenicity, and is big to the healing and the influence of healing back of wound.It is to use collagen and the dressing of the synthetic collagen class of other material that extracts from heterogenous animal skin that modern times are used more dressing clinically.
Collagen is a kind of native protein, extensively is present in skin, tendon and other connective tissue of animal.Collagen be epidermis cell migration, propagation place mat support, and provide good Basal nutrition, help epithelial propagation reparation, thereby can promote the healing of wound surface.As far back as the seventies.Cover the clinical report of burn wound with regard to useful collagem membrane.Wherein Yang has developed a kind of YCWM collagem membrane, shows by clinical trial, and this film is easy to use, to infect, the therapeutic effect of exsiccant burn and skin donor site wound surface is remarkable.Usefulness gastric enzyme digestion methods such as Gao extract collagen from Corii Sus domestica corium, adopt salting out method to make collagem membrane, and carried out clinical experimental study with this collagem membrane, and the result shows that this film has tangible promotion wound healing ability.The eighties, China was studied in the application of burn wound collagen protein.Third Military Medical University extracts Collagen Type VI from pig leather, and wiring solution-forming is used for the treatment of deep burn wound and remaining granulation wound, and not only wound healing is fast, and more back skin is smooth.Nineteen ninety, first Affiliated Hospital of The 2nd Army Medical College made collagen by oneself with fresh porcine skin, treatment burn back residual wound 8 examples, and the result shows that collagen can obviously promote wound healing.Western countries such as the U.S. in 1994, Italy, Germany have begun to carry out the phase clinical stage.
Since collagen by with platelet adhesion, aggregation, thereby promote the hemagglutinative function of wound.Zoopery and the clinical experiments a large amount of as far back as the end of the sixties have just confirmed that collagen has excellent anthemorrhagic performance.The beginning of the nineties from the collagen sponge of U.S.'s import at home part hospital carried out clinical verification.The result shows that the more traditional gelfoam of collagen sponge has better anthemorrhagic performance.
In sum, collagen has good promotion wound healing and anthemorrhagic performance, is surgical dressing material preferably, but at present both at home and abroad employed all be the collagem membrane of pure system, this glued membrane complex manufacturing process, waste of raw materials is serious.And be not easy to sterilization and preserve, bring certain difficulty for simultaneously medical worker's dressing change.
The object of the present invention is to provide a kind of preparation method of new pattern compress; be primarily characterized in that: possess nontoxic, nonirritant and antigenicity, be convenient to clinical practice; possess good toughness, water penetration and microorganism buffer action; possess the protection cambium simultaneously and keep wound surface humidity and temperature, promote the effect of wound healing.Possess hemostasis and bacteriostasis.Possess the protection cambium and safeguard wound surface and the favourable wound healing of temperature, and this dressing preparation technology is simple, raw material sources are extensive.
Purpose of the present invention can reach by following technical measures: select corium and the skin histology of healthy mammal for use, add water, adopt any machinery to smash to pieces, centrifugal or other any method is separated upward cleer and peaceful sediment; Supernatant is used to prepare the growth activity factor, and sediment is used to prepare collagen.Adopt the supernatant that extracts, cross malleation and hold back post, get filtrate, degerming claims No. 1 liquid.Sediment dissolves with diluted acid, with rare NaOH adjust pH to 7.0, gets filtrate after the filtration, and degerming claims No. 2 liquid.Mix above-mentioned two liquid by proper proportion, promptly biological raw material.The support dressing autoclaving of going ahead of the rest adds an amount of biological raw material again, low temperature or any temperature drying, and through Physical Processing, packing, end product.
In such scheme, healthy mammal is selected from as pig, cattle, horse, donkey, sheep, Canis familiaris L., rabbit etc.
In such scheme, support dressing can select for use traditional gauze dressing and other to possess the dressing of good toughness, breathability, water penetration and microorganism buffer action as cotton, line, yarn, cloth and synthetic fibers etc.
In such scheme, the ratio that preferably adds dilute hydrochloric acid is that every 1L sediment adds the 2L dilute hydrochloric acid, and addition can be from 1L to 5L.
In such scheme, preferred baking temperature is-10 ℃, vacuum drying, and the time presses degree of drying and determines.Other temperature can not wait from-10 ℃-+95 ℃, and the time presses the degree of drying difference and do not wait from one hour to tens of hours.
Below, will be described in further detail the present invention with embodiment.
Embodiment 1
Select corium and the skin histology of healthy piglets for use, add water, adopt any machinery to smash to pieces, even oar, centrifugal or other any method is separated upward cleer and peaceful sediment; Supernatant is used to prepare the growth activity factor, and sediment is used to prepare collagen.Adopt the supernatant that extracts, cross malleation and hold back post (is 50,000 by molecular weight), get filtrate, degerming claims No. 1 liquid.The 1L sediment with rare NaOH (1mol/L) adjust pH to 7.0, is got filtrate with 2L dilute hydrochloric acid (0.1mol/L) dissolving after the filtration, degerming claims No. 2 liquid.Mix above-mentioned two liquid by proper proportion, promptly biological raw material.The support dressing autoclaving of going ahead of the rest adds an amount of biological raw material liquid again ,-10 ℃ of vacuum dryings, and through Physical Processing, packing, end product.
Embodiment 2
Select corium and the skin histology of healthy piglets for use, add water, adopt any machinery to smash to pieces, homogenate, centrifugal or other any method is separated upward cleer and peaceful sediment; Supernatant is used to prepare the growth activity factor, and sediment is used to prepare collagen.Adopt the supernatant that extracts, cross malleation and hold back post (is 100,000 by molecular weight), get filtrate, degerming claims No. 1 liquid.The 1L sediment with rare NaOH (1mol/L) adjust pH to 7.0, is got filtrate with 4L dilute hydrochloric acid (0.1mol/L) after the filtration, degerming claims No. 2 liquid.Mix above-mentioned two liquid by proper proportion, promptly biological raw material.The support dressing autoclaving of going ahead of the rest adds an amount of biological raw material liquid again, 80 ℃ of vacuum dryings, and through Physical Processing, packing, end product.
Embodiment 3
Adopt this method can observe No. 1 liquid and promote the fibroblasts proliferation activity.
Material and method
1. fibroblastic separation: get the piglets adrenal gland, shred, add 0.25% pancreatin, digestion is 30-60 minute under the room temperature; The individual cells of separate out suspended, centrifugal, remove pancreatin, cultivating the keynote cell concentration with the DME/F12 that contains 10% calf serum is 5-10 * 10
8/ L; Cultivated 2 hours with culture bottle, remove supernatant, add the DME/F12 culture medium that contains 10% calf serum and continue to cultivate, wait cell to cover with after, use the trypsinization attached cell, do active determination test after the accent cell concentration changes liquid.
2. Determination of biological activity: the accent cell concentration is 4.0-8.0 * 10
8/ L is inoculated in 96 well culture plates, the 0.1ml/ hole.Put 37 ℃ of 5%CO
2Change liquid after 24 hours, add No. 1 liquid of 25-1000mg/L, with 20 meters positive contrasts of g/L EGF, with the negative control wells of normal saline.Abandon supernatant after 72 hours.96 orifice plates add and contain 0.15g/L MTT and 2mci/L
3H-TDr F
12Culture fluid 0.05ml.Cultivated 4 hours, do respectively the MTT colorimetric and
3The technical operation that H-TDr mixes.
3. statistical procedures adopts t check or variance test.
The result
1.1 number liquid is to the influence of fibroblast proliferation
(1) No. 1 liquid is to cultivating the influence of 72 hours rat fibroblast propagation.
No. 1 liquid is to fibroblast
3The influence that H-TdR mixes (X ± SD, n=8)
| Concentration (mg/L) | Cpm(10 -1) | Handle number | The t value |
| 1,000 500 250 100 50 25 EGF control groups | 1194±152 1805±280 2755±431 3506±1302 3019±1381 2713±834 3135±727 1641±334 | 1 1 3 6 4 2 5 | 1.540 0.565 3.839* 6.427** 4.749** 3.695** 5.149** |
| The F value | 7.215 |
Annotate: *, P＜0.05, * *, P＜0.01, with matched group relatively.
(2) No. 1 liquid are to cultivating the influence of 72 hours rat fibroblast vigor.
No. 1 liquid to the influence of fibroblast dehydrogenase activity (X ± SD, n=8)
| Concentration (mg/L) | Cpm(10 -1) | Handle number | The t value |
| 1000 500 250 100 50 25 EGF | 0.190±0.0346 0.227±0.0580 0.373±0.0763 0.307±0.0230 0.280±0.0361 0.297±0.0763 0.372±0.0627 | 1 1 6 4 2 3 5 | 1.237 0.397 6.846** 3.931** 2.738* 3.180* 6.801** |
| Matched group | 0.218±0.0356 |
| The F value | 11.297 |
Annotate: *, P＜0.05, * *, P＜0.01, with matched group relatively.
Conclusion
No. 1 liquid can obviously promote the vigor of the synthetic and dehydrogenase of the fibroblastic DNA of adrenal gland, and is certain dosage action effect; Its optimum concentration is 100mg/L, and optimum efficiency is similar with 20 meters g/LEGF effects.
Embodiment 4-5
The efficacy analysis judgement of this dressing is carried out from zoopery and clinical research two aspects respectively.Concrete parameters for observation on effect is as follows:
1. safety criterion: observe the full course of treatment
1. general inspection: whole body or local untoward reaction
2. hematuria routine
3. hepatic and renal function
2. wound surface situation
1. outward appearance, edge of wound react, ooze out situation, infection conditions
2. wound healing: healing area percentage, healing rate, the wound surface degree of depth and the complete healing time of wound surface
3. different time wound tissue specimen pathological examination results
4. granulation growing state in the different wound surface of different time
5. quality after wound surface is healed
Curative effect judging standard and foundation thereof
1. with time healing wound surface percentage ratio
2. with the time wound surface degree of depth
3. the complete healing time of wound surface
4. wound surface granulation growing state
5. pathological examination
(zooperal measurement data adopts pairing T check; The measurement data of clinical experiment adopts the T check of non-matching)
Embodiment 4
This dressing influences the healing of wound surface by a plurality of links.We are to make children pig in age by oneself, and the bark fetching wound surface is an animal model, relatively makes the influence to wound healing of biological dressing and traditional kpetrolatum gauze by oneself.
Materials and methods
Material: 5 of 2-3 monthly age pigletss, male and female are not limit, and body weight 15-20kg is provided by the Agricultural University Of South China animal.The aseptic kpetrolatum gauze dressing of tradition.The self-control biological dressing.The routine operation apparatus.
Method:
The animal model preparation: cut 5 area sizes every laboratory animal spinal column both sides and be 5cm * 5cm, the degree of depth is the foursquare symmetry wound surface of 1.5mm (thick intermediate thickness free skin graft).
Research method: contrast takes consubstantiality, antimere, be the biological dressing experimental group with 5 wound surface in left side of an animal promptly with degree of depth wound surface, and 5 wound surface in right side are traditional kpetrolatum gauze matched group.Influence wound healing for anti-, we changed dressings every 3 days, and observed and recorded wound healing situation, wound healing rate are changed dressings till wound healing, and performed record simultaneously.Cut the tissue specimen in each wound surface of experiment and matched group at every turn when changing dressings at random, and send pathologic finding.
Experimental result
1. wound surface area change (CM
2)
| Time (d) | 1 | 4 | 7 | 11 | 14 | 17 |
| The experimental group matched group | 25 25 | *11.3±1.42 18.73±0.98 | *5.15±0.87 12.4±1.22 | * fully recover 7.64 ± 0.52 | 2.13±0.23 | Recovery from illness |
* P＜0.05, the vs matched group.
2. complete healing time
Experimental group average out to 10.24 ± 1.12 days
Matched group average out to 15.87 ± 0.94 days
3. the experimental group edge of wound is red, swollen obviously light than matched group.
4. experimental group granulation growing state is good, granulation quality aquatic foods, and toner is red, smooth surface, the granulation granule is evenly fine and smooth, oozes out to lack, and inflammatory reaction is light.Matched group granulation growing state is compared with experimental group: the granulation quality is coarse, and colour cast is white, and the granulation granular size is inconsistent, oozes out morely relatively, and inflammatory reaction is heavier.
5. healing skin situation:
Experimental group sees that wound surface does not stay cicatrix, and skin colour and normal skin are very nearly the same, and the wound surface skin elasticity is good, the quality softness.Hair growth is good.
Matched group sees that there is the tiny cicatrix of being dispersed in property in wound surface, and skin retains bolarious pigmentation spots, and the wound surface skin elasticity is not good enough, and quality is hard.Hair growth is relatively poor.
6. the check pathological section result shows:
Experimental group: the postoperative focal holostrome necrosis of epidermis in the 4th day, corium and subcutaneous tissue have indivedual inflammatory cell infiltrations; The 7th day epidermis of performing the operation is roughly normal, corium and subcutaneous fibroblast hypertrophy and the inflammatory cell infiltration of as seen being dispersed in, epidermis hyperplasia of squamous epithelium keratinization; The 11 day slight keratinization of epidermis of postoperative, corium and subcutaneously see indivedual inflammatory cell infiltrations.
Matched group: the 4th day interior visible microabscess of epidermis of postoperative forms, corium and subcutaneous a large amount of fibroblast hypertrophy, and more inflammatory cell infiltration, and see that the epithelioid cell tuberosity forms; Postoperative was seen fibrofatty tissue, focal inflammation cellular infiltration on the 7th day; Postoperative was seen part not exclusively epidermis, corium and subcutaneous a large amount of proliferation of fibrous tissue on the 11st day, and more inflammatory cell infiltration was arranged, epidermal hyperplasia keratinization, corium and subcutaneous a large amount of proliferation of fibrous tissue.
Embodiment 5
The clinical experiment case is included standard in: the patient is voluntary; There are not the heart of merging, liver, kidney and serious pathological changes of hemopoietic system and spiritual patient; Burn or other traumatic cicatrix are cut the patient that need make skin graft after the scar.
Materials and methods
Test material: self-control biological dressing; Petrolatum oil gauze
Method: contrast takes the same position of Different Individual, with degree of depth skin donor site wound surface, changes dressings once in per 2 days, changes dressings all with condition cleaning wound surface at every turn, all observes when changing dressings at every turn and writes down the wound surface area, heals fully until wound surface.Above therapeutic operation is born by same people.
Experimental result
One, physical data
(1) case source: this experiment case is cut the patient of scar skin-grafting institute's Burn and Plastic Surgery Dept. 6-12 month in 1998 in hospital for me, and totally 38 examples are assigned randomly in experimental group and the matched group.Wherein experimental group and matched group are 19 routine patients.
(2) sex and age see the following form
| The man | The woman | Age (X ± SD) |
| 21 | 17 | 24±9 |
(3) skin donor site wound surface area (cm
2) and the degree of depth: the experimental group average area is 96.53 ± 14.23; The matched group average area is 92.96 ± 15.41.Each case skin donor site is the split thickness skin graft wound surface.
Two, efficacy analysis
(1), ordinary circumstance: experimental group has matched group hematuria routine, hepatic and renal function there is no to cause abnormal change before and after the treatment; Do not see whole body and local anaphylaxis and other untoward reaction in the therapeutic process.
(2) red, the swollen reaction matched group of matched group edge of wound is obviously serious than experimental group.
(3) granulation tissue growing state:
Experimental group granulation growing state is good, granulation quality aquatic foods, and toner is red, smooth surface, the granulation granule is evenly fine and smooth, oozes out to lack, and inflammatory reaction is light.Matched group granulation growing state is compared with experimental group: the granulation quality is coarse, and colour cast is white, and the granulation granular size is inconsistent, oozes out morely relatively, and inflammatory reaction is heavier.
(4) wound surface area change
| Time (d) | Experimental group (cm 2) | Matched group (cm 2) |
| 1 3 5 7 9 11 13 14 16 17 19 21 | 96.53 ± 14.23 87.32 ± 9.25 * 65.60 ± 17.21 * 52.72 ± 2.78 * 39.71 ± 19.86 * 20.83 ± 4.40 14.69 ± 3.01 5.11 ± 1.20 * recovery from illness | 92.96 ± 15.41 91.44 ± 11.34 83.45 ± 9.53 71.70 ± 21.22 59.49 ± 8.65 47.52 ± 9.07 30.26 ± 4.31 17.91 ± 5.54 8.39 ± 0.92 2.75 ± 1.95 0.97 ± 0.07 recoveries from illness |
* P＜0.05, the vs matched group
(5) natural law that heals fully:
Experimental group is 14.74 ± 2.37;
Experimental group is 19.23 ± 3.82.
(6) experimental group sees that wound surface does not stay cicatrix, and skin colour and normal skin are very nearly the same, and the wound surface skin elasticity is good, the quality softness.
Matched group sees that there is the tiny cicatrix of being dispersed in property in wound surface, and skin retains bolarious pigmentation spots, and the wound surface skin elasticity is not good enough, and quality is hard.
(7) pathologic finding
Roughly similar with the zoopery part
Result and discussion
Wound repair needs a good gnotobasis, and dressing is to safeguard surface of a wound gnotobasis and protection or provide surface of a wound part to be conducive to the microenvironment of Wound healing---comprise certain temperature, humidity, acid-base value and various nutrition. So the dressing of ideal type should possess the extraneous bacterium of isolation, suppress the characteristic of bacterium in the wound; Also should possess the key properties such as the tissue repair of assurance and wound healing. This dressing is to be formed by normal gauze and the sucking pig bark extract system of closing, it has the normal gauze isolation, adsorption and permeation is good, cheap and characteristics easy to use, the advantage that has simultaneously collagen and little molecule prognosis, can prevent that too much water evaporates from losing, keep temperature, the humidity of the surface of a wound, the most important thing is that it has the effect of Promote cell's growth and wound healing.
Analyze in zoopery and clinical and experimental study data from us: the effect of homemade biological dressing wound healing is obviously good than control group, and the wound healing quality is also obviously good than control group. After using homemade biological dressing, the edge of wound inflammatory reaction is light than control group, illustrates that this dressing also has antibiotic or antibacterial effect. Have no toxic and side effect or bad reaction in the application process.
Claims (6)
1, it is raw material that a kind of preparation method of biological dressing, this preparation method are selected corium of health pig childhood, cattle, horse, donkey, sheep, Canis familiaris L., rabbit and skin histology for use, adds water, and machinery is smashed to pieces, and centrifugalize goes out supernatant and sediment; Supernatant is crossed malleation and is held back post, gets filtrate, and degerming gets No. 1 liquid; Sediment dissolves with 1-5 times of volume diluted acid, with dilute NaOH solution adjust pH to 7.0, gets filtrate after the filtration, and degerming gets No. 2 liquid; Mix above-mentioned two liquid, get biological raw material; Add biological raw material behind the support dressing autoclaving, drying, processing, packing gets finished product at last.
2, preparation method according to claim 1 is characterized in that described drying carries out under-10 ℃～95 ℃, the time is 1-10 hour.
3, preparation method according to claim 1 and 2 is characterized in that described drying is under-10 ℃, carries out vacuum drying.
4, preparation method according to claim 1 and 2 is characterized in that described diluted acid is the hydrochloric acid of 0.1mol/L.
5, preparation method according to claim 1 and 2 is characterized in that described dilute NaOH solution is 1mol/L.
6, preparation method according to claim 1 and 2 is characterized in that described support dressing is gauze dressing or cotton, line, yarn, cloth and synthetic fibers dressing.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB991077741A CN1213774C (en) | 1999-05-28 | 1999-05-28 | Technology for preparation of biological dressing material |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB991077741A CN1213774C (en) | 1999-05-28 | 1999-05-28 | Technology for preparation of biological dressing material |
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| CN1275408A CN1275408A (en) | 2000-12-06 |
| CN1213774C true CN1213774C (en) | 2005-08-10 |
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| CNB991077741A Expired - Fee Related CN1213774C (en) | 1999-05-28 | 1999-05-28 | Technology for preparation of biological dressing material |
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| GB0809592D0 (en) * | 2008-05-27 | 2008-07-02 | Imp Innovations Ltd | Biomaterials |
| CN101507828B (en) * | 2009-03-20 | 2012-06-06 | 武汉锐尔生物科技有限公司 | Dressing production process |
| CN105903058B (en) * | 2016-05-30 | 2020-11-10 | 山西大学 | Preparation method of transparent film band-aid containing traditional Chinese medicine components |
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