CN1274370C - Active cell-less corium ground substance - Google Patents
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- CN1274370C CN1274370C CN 02112455 CN02112455A CN1274370C CN 1274370 C CN1274370 C CN 1274370C CN 02112455 CN02112455 CN 02112455 CN 02112455 A CN02112455 A CN 02112455A CN 1274370 C CN1274370 C CN 1274370C
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Abstract
The present invention provides an activated cell-free dermal matrix used for skin transplantation and a preparation method thereof. The matrix comprises a dermal matrix and an effective quantity of cell factors, wherein the dermal matrix does not comprise cells basically; the effective quantity of cell factors are selected from the following group: fibroblast growth factors, vascular endothelial growth factors, platelet derived growth factors and a transforming growth factor-alpha or the mixture of the group. Favorable blood circulation can be established between the activated cell-free dermal matrix of the present invention and a wound surface substrate in a short time. The successful rate of transplantation is increased, and the effect of transplantation is improved.
Description
Technical field
The present invention relates to medical domain, relate more specifically to be used for dermatoplastic active cell-less corium ground substance and preparation method thereof.
Background technology
The modern treatment of holostrome skin injury wound surface is still thick in body skin (being thin skin) to transplant sword.Because sword is thick to contain less corium composition from the body skin, can produce scar hyperplasia in various degree behind the wound healing.If transplant thicklyer, then often cause tangible scar hyperplasia at skin donor site from the body skin.
People such as Bell utilized the natural collagen protein of extraction to mix according to a certain percentage with fibroblast in 1981, formed a skin corium.Again epidermal cell suspension is seeded on this skin corium, makes the Composite Skin of a similar normal skin.
Hznsbrough also utilizes the substrate framework of biodegradable polymer as the cell growth, external the neonate fibroblast is inoculated in the substrate framework.Along with the growth of cell on framework, they constantly secrete compositions such as numerous protein and glycoprotein, are filled in the grid framework, form a natural extracellular matrix.Show that through experiment this dermal substitute with vital activity covers vascularization rapidly on the degree of depth wound surface, and covering thereon from body surface skin also epithelization thereupon.
Heck is in report in 1985, with allogeneic dermis as from the transplanting carrier of body surface skin and be applied to rat and human body on.He adopts the natural allogeneic skin crossed through freezing processing by the vacuum attraction method gained to be covered allosome corium surface from the body skin.
Some above-mentioned composite transplantation methods, part is set up the 26S Proteasome Structure and Function with similar corium.It is smooth, smooth that the skin-grafting area wound surface becomes; Reduced scar hyperplasia, more the back function is also comparatively perfect.
The monolayer epidermis cell of people such as Cuono after by In vitro culture is covered in the allosome corium surface of removing epidermis.Though Preliminary Applications is in clinical for this method, effect is difficult to certainly.Though being this method, reason removed epidermis, thereby reduced immunizing antigen, can not cause acute rejection, but owing to kept epidermis cell composition, fibroblast and vascular endothelial cell etc. that skin corium contains, they still can cause body and produce immunological rejection.
Nineteen ninety-five U.S. Lifecell Corp. Livesey[Livesey SA, Herndon DN, Hollyoak MA, et al:Transplanted Acellular Allograft Dermal Matrix.Transplantation, 1995,60 (l): 1～9] etc. at first reported the preparation method of cell-less corium ground substance.The same year the U.S. Wainwright[Wainwright DJ:Use of an Acellular Allograft Dermal Matrix (AlloDerm) in the Management of Full-thickness Burns.Burns, 1995,21 (4): 243～248] at first report the clinical practice of cell-less corium ground substance, caused the extensive attention of international burn educational circles; Domestic many units have also carried out clinical practice in succession, and its range of application expands the plastic surgery to, and neck face surgery etc. have been obtained clinical effectiveness preferably.
(Acellular Dermal Matrix is a kind of dermal substitute that is used for holostrome skin injury wound surface ADM) to cell-less corium ground substance, derives from natural skin.It is after natural skin is handled through certain physico-chemical method, to have removed all cells composition in epidermis and the corium, but kept collagen composition and its three-D space structure of corium, has also kept the basement membrane complex simultaneously.Owing to removed the cell component of tool strong antigen in the skin, and extracellular matrix components such as the faint antigenic collagen protein of only residual tool, simultaneously because it has natural dermis, can induce the cell component of transplanting the back host (as fibroblast, endotheliocyte etc.) follow its supporting structure growth, dermal matrix also can be supported fibroblastic infiltration, new vessels forms, make fibroblast is synthetic and secretion has normal configuration and function extracellular matrix such as collagen, make the regeneration of granulation tissue fast-ripenin and corium deep layer, being reduced to fibrocyte breaks up to myofibroblast, and with its on transplant merge from body surface skin composition, form comparatively complete skin texture, substantially recovered the original function of skin, thereby produced good few scar or do not had the clinical effectiveness of scar.Cell-less corium ground substance be that treatment at present needs up-and-coming method in the holostrome skin injury of skin-grafting from body surface skin cograft.
Yet, the outstanding problem that runs in the cell-less corium ground substance application process is: after cell-less corium ground substance is transplanted, in the short time (3～5 days), as fail to set up good blood circulation with the wound surface substrate, then can cause serious adverse consequences, show as: (1) graft anti-infection ability is poor, easily causes graft failure because of taking place to infect; (2) fail to provide enough nutrition from the body surface skin, thereby influence the latter's survival for what transplant on it.
Therefore, this area presses for provides new, effectively treat the graft of holostrome skin injury wound surface.
Summary of the invention
Purpose of the present invention just provides a kind of graft and method for making thereof of effective treatment holostrome skin injury wound surface newly.
In a first aspect of the present invention, a kind of graft is provided, it comprises:
(a) dermal matrix, described dermal matrix is substantially free of cell;
(b) cytokine of group under being selected from of effective dose: fibroblast growth factor, vascular endothelial cell growth factor, platelet derived growth factor, transforminggrowthfactor-or its mixture.
In a preference, described dermal matrix also contains the basement membrane complex.
In another preference, cell quantity preferably less than 1%, more preferably less than 0.5%, best less than 0.1%, is pressed the stereometer of dermal matrix less than 5% in the described dermal matrix.
In another preference, described cytokine is selected from: fibroblast growth factor, vascular endothelial cell growth factor or its mixture.
In another preference, described effective dose is the 100-30000AU/ cubic centimetre.
In another preference, the thickness of described dermal matrix is 0.2-2mm, and described cytokine is a basic fibroblast growth factor.
In another preference, described dermal matrix is from the skin of following animal: pig, cattle, people, Mus.
In a second aspect of the present invention, a kind of method for preparing graft is provided, comprise step:
(a) provide a dermal matrix, be substantially free of cell in the described dermal matrix;
(b) dermal matrix is contacted with the cytokine that is selected from down group, contain the described cytokine that concentration is the 100-30000AU/ cubic centimetre in the dermal matrix thereby make: fibroblast growth factor, vascular endothelial cell growth factor, platelet derived growth factor, transforminggrowthfactor-or its mixture.
In a preference, in step (b), with the solution injection dermal matrix of needleless, high pressure filling gun with described cytokine; And the concentration of described solution is 240-24000AU/ml or 0.1-10ng/ml.
In in another preference, described cytokine is a basic fibroblast growth factor.
In a third aspect of the present invention, a kind of test kit is provided, it contains graft of the present invention and optional other reagent and description.
The specific embodiment
Problem at the existence of above-mentioned this area, the inventor is extensive studies through going deep into, discovery is by adding the somatomedin that can promote angiogenesis, set up good blood circulation with the wound surface substrate at short notice after can impelling cell-less corium ground substance to transplant, improve transplanting succeed rate, and improve its transplantation effect.Finished the present invention on this basis.
Term
Term " purification or isolating " refers to purification or isolating material is substantially devoid of other cells, protein or polypeptide, for example the cytokine of purification or the isolated cells factor.
Term " xenotransplantation " refers to required biomaterial (as skin) is taken out and is applied to the method for another species object from a certain species.
Term " autotransplantation " refers to required biomaterial (as skin) is taken out and is applied to same patient's method from certain patient.
Term " heteroplastic transplantation " refers to required biomaterial (as skin) is taken out and is applied to another different patients' method from certain patient.
Term " isograft " refers to the cell transplantation that takes place between people identical in the heredity.
Cell-less corium ground substance
Can be used for cell-less corium ground substance of the present invention and be not particularly limited, can be with mammiferous natural skins such as source people, pig, cattle, by the cell-less corium ground substance of known method preparation.
With regard to the main component of cell-less corium ground substance, for having removed epidermis composition and all cells composition in the skin really in the normal skin, and kept the collagen composition in the corium and its three-D space structure, preferably also kept the basement membrane complex simultaneously.Collagen in the cell-less corium ground substance is the III Collagen Type VI that is present in the substrate, is present in III type, VII Collagen Type VI in the basement membrane.In addition, also have elastic fibers, chondroitin sulfate, fibronectin, aminoglycan in the substrate.
In dermal matrix of the present invention, cell quantity preferably less than 1%, more preferably less than 0.5%, best less than 0.1%, is pressed the stereometer of dermal matrix less than 5%.A kind of easy detection method is that cell counting is less than 3, preferably less than 2, more preferably less than 1 when optical microscope high power field (* 40) is observed down.
A kind of preferred dermal matrix also contains the basement membrane complex, and its effect is the connection of strengthening between corium and the epidermis, and improves the quality after the skin graft healing.
The thickness of cell-less corium ground substance is not particularly limited, and is about 0.2-2mm usually, more preferably is about 0.30-0.50mm.
The size of cell-less corium ground substance is not particularly limited, and can make according to wound surface is different, also can be made into for example specific standard of 100mm * 100mm, then the dermal matrix of required size under the cutting in use.
Promote the cytokine of angiogenesis
As used herein, term " factor or the cytokine of promotion angiogenesis " refers to angiogenesis is had the material of promotion or activation, mainly comprise: fibroblast growth factor (fibroblast growth factor, FGF), vascular endothelial cell growth factor (vascular endothelial growth factor, VEGF), platelet derived growth factor (Platelet-derived growth factor, PDGF), transforming growth factor-o (transforming growth factor-α, TGF-α) etc.Preferred cytokine is FGF (comprising basic fibroblast growth factor bFGF) and VEGF, and that best is fibroblast growth factor bFGF.
The regulation and control that the VEGF vascular endothelial cell growth factor is considered to act on the short endothelial cell proliferation the strongest, that specificity is the highest, migration because of in.In the wound healing process, VEGF promotes the wound surface vascularization; Only find that so far vascular endothelial cell to the breeder reaction that VEGF has high specific, yet there are no the report of other cell to the VEGF reaction.Therefore, what of VEGF are closely related with vascularization in the wound tissue.In addition, VEGF participates in regulating vascular endothelial cell (endothelial cell, the formation of EC) migration, propagation and pipe spline structure.The VEGF deficiency can cause lumen of vessels closure, blood vessel to be degenerated.
Basic fibroblast growth factor (bFGF) is a kind of very polypeptides matter of trace that exists in mammal and the human body, embryo's layer and the cell in outer embryo's layer source such as the division and the propagation of fibroblast, vascular endothelial cell etc. in stimulating, physiological function is very extensive.BFGF is to the three phases of processes of wound repair, and promptly all there are significant facilitation in the local inflammation stage of reaction, cell proliferation and differentiation and granulation tissue formation stage, tissue reconstruction stage.In cell proliferation and reparation phase, one of step of most critical is the formation of granulation tissue, the essence of granulation tissue is a large amount of blood capillary and abundant fibroblasts, bFGF is the efficient growth promoter of fibroblast, vascular endothelial cell and vascular smooth muscle cell just, formation that can the intense stimulus new capillary vessel, significantly increase granulation tissue blood capillary quantity and blood flow, improve the wound surface microcirculation, for tissue repair provides necessary oxygen and rich nutrient substances.So the formation of new vessels is closely related in the content of wound surface bFGF and the wound healing.
Utilize this biological agent of somatomedin such as bFGF and VEGF, can improve the survival rate after cell-less corium ground substance is transplanted, and then improve its transplantation effect.The VEGF source is limited, costs an arm and a leg, and has limited its application.
More than past ten years, biology and medical worker have carried out extensive and deep basic and applied research to bFGF both at home and abroad.Developed countries such as the U.S., Japan, France quantity research and the producer gene recombinant bfgf of measuring one's own ability that all have high input; The China national State Scientific and Technological Commission also lists the research of bFGF in " eight or five " and " 95 " brainstorm project in succession.1998, the bFGF bovine basic fibroblast growth factor (rb-bFGF) of China's development obtained the formal production certification that Ministry of Public Health is issued, and indicates that bFGF has finished the course from the laboratory to the industrialization finally.
The source that can be used for each cytokine of the present invention is not particularly limited, and can use natural, isolating, reorganization or synthetic cytokine, and separate, technology recombinant expressed and/or the various cytokines of synthetic is as known in the art.In addition, also be commercially available each cytokine, for example bFGF can be available from east, Zhuhai east mcroorganism engineering company of large group.
Other additives
In active cell-less corium ground substance of the present invention; except the cytokine that contains above-mentioned promotion angiogenesis; can also contain some other additive, for example prevent the wound infection inflammation antibiotic, prevent the protective agent (as heparin) of somatomedin degraded.
Preparation method
After the cytokine that has obtained cell-less corium ground substance and purification, dermal matrix is contacted with cytokine, thereby make the cytokine that contains effective dose in the dermal matrix.As used herein, refer to the amount of needed multiplicaiton factor with the term " effective dose " of cytokine logotype for the cell proliferation of realizing desired level.The effective dose of specific growth factor depends on various variables, comprising, the proliferating cells type is used and treated to required propagation level, certain factor whether with other factors.For example, for bFGF, its effective dose is generally the cubic centimetre into 100-30000AU/, preferably 200-10000AU/ cubic centimetre, more preferably 500-7500AU/ cubic centimetre, 1000-5000AU/ cubic centimetre best.
In the present invention, preparation method is not particularly limited, if this method can make cytokine be evenly distributed among the dermal matrix and or on.For example, cell-less corium ground substance is placed (hatching) a period of time in the solution of factor-containing.
It is particularly preferred that two kinds of preparation methoies are arranged.First kind is will promote the somatomedin of angiogenesis to inject cell-less corium ground substance with needleless, high pressure filling gun (for example can be available from MadaJet XL, the needleless, high pressure filling gun of USA).The advantage of this method is to utilize pressure that cytokine is penetrated among the dermal matrix equably, and can keep certain hour (about 24-48 hour or longer).
Second method is the somatomedin that adds certain density promotion angiogenesis at cell-less corium ground substance and wound surface substrate intersection.The advantage of this method is simple and convenient, shortcoming be somatomedin difficulty infiltrate through in the dermal matrix and the weak point of holding time.
These two kinds of methods can both be impelled grow into cell-less corium ground substance and form blood capillary of endotheliocyte, thereby thereby improve the cell-less corium ground substance transplanting succeed rate and improve its transplantation effect.
The detection of wound healing process medium vesselsization can be undertaken by detecting following index.
1) the VIII factor claims christmas factor or VWF again.Vascular endothelial cell can be secreted and discharge the VIII factor, and the VIII on the VIII factor antibody that has a label and the endothelial cellular membrane by chromogenic reaction, develops the color vascular endothelial cell because of combining in antigenic specificity.
2) CD31 PECAM (Platelet endothelial cell adhesionmolecule).CD31 is that a kind of 130Kda integrates mucosa albumen, belongs to ig supergene family, mediated cell and cell adhesion.CD31 is continuous expression in endotheliocyte, therefore can be used as the vascularization sign.
3) CD34, in the endotheliocyte adhesion process, playing the part of has the key player, is angiopoietic important symbol.Experimentation shows that at the newborn blood vessel that generates of wound surface, its CD34 expresses generally to be increased, the fine labelling endotheliocyte of anti-CD34 antibody capable, and can distinguish new vessels and normal structure blood vessel.
But above-mentioned these index applied immunologies and histochemistry principle detect, and the antigen that wound tissue section medium vessels endotheliocyte is had develops the color, utilize optical microscope that the blood capillary in the wound tissue is counted, thereby draw the microvessel density in the wound tissue, the vascularization degree of reflection wound surface.
Experiment of the present invention shows, use active cell-less corium ground substance of the present invention after, set up good blood circulation with the wound surface substrate at short notice after impelling cell-less corium ground substance to transplant, improve transplanting succeed rate, thereby improve its transplantation effect.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
Embodiment 1
The preparation of active cell-less corium ground substance
In this embodiment, prepare active cell-less corium ground substance by following steps, following steps all require the sterile working.
Step 1: cleaning level SD rat, losing hair or feathers back 24 hours in the back, cuts the full thick skin in back (about 15 * 20 square centimeters of size); Instead on bark fetching drum get middle pachydermia; 1. go epidermis to handle: add 2.5 units/ml Bacillus polymyxa Neutral proteinase II (Dispase II, Roche company), effect is 24 hours under 4 ℃ of conditions, finishes the back in effect and removes epidermis with the elbow skin forceps, and inhale and remove epidermis liquid.2. taking off cell handles: add 0.5% sodium lauryl sulphate (SDS is called for short detergent) 160ml, 20～26 ℃ act on 24 hours; Absorb detergent, last with Hanks liquid flushing twice, absorb Hanks liquid, can obtain cell-less corium ground substance.Be placed on then in the Hanks liquid that contains penicillin (100u/ml), chloromycetin (100 μ g/ml) and kanamycin (100 μ g/ml), 4 ℃ of preservations are standby.
Qualified cell-less corium ground substance meets following requirement: removed all cells composition in epidermis and the corium, comprise complete cell and cell debris (cell debris≤1/10 high power field), but the three-D space structure that has kept corium has kept the basement membrane complex.
Step 2: contain 5 milliliters 240,2400, the bFGF solution of 24000AU/ml (0.1,1,10ng/ml), (available from MadaJet XL, USA) inject cell-less corium ground substance, obtain containing the bFGF cell-less corium ground substance with the needleless, high pressure filling gun.The concentration of bFGF is about the 150AU/ cubic centimetre respectively, 1500AU/ cubic centimetre and 15000AU/ cubic centimetre in the active cell-less corium ground substance that obtains.
Embodiment 2
The animal experiment of active cell-less corium ground substance
In this embodiment, utilize zoopery to test.Method of testing is as follows: selecting the cleaning level SD rat of some, lost hair or feathers back 24 hours in the back, cut a certain size holostrome skin at people Mus back with scalpel, and the anti-sword pachydermia of getting on the drum-type dermatome is as the lean type skin.Wound surface is divided into three groups at random:
Organize 1 wound surface as the experiment wound surface, after the hemostasis, be implanted among the embodiment 1 active cell-less corium ground substance of preparation earlier, autologous transplanting skin thereon then, pressure dressing;
Organize 2 wound surface as the experiment wound surface, after the hemostasis, adding concentration is the bFGF (volume is the 5-10 milliliter) of 240～24000AU/ml or 0.1～10ng/ml, successively transplants cell-less corium ground substance then and from body skin, pressure dressing;
Organize 3 wound surface wound surface in contrast, after the hemostasis, do not add bFGF, but the same value of moving cell-less corium ground substance successively and from body skin, pressure dressing.
After two weeks, above-mentioned every treated animal is all put to death in the back of drawing materials.Every treated animal is divided into two groups more at random when drawing materials.The first little treated animal is opened wound surface, gets specimen, and its paraffin section immunohistochemistry detects the VIII factor (the vonWillebrand factor), CD31 and CD34; The second little treated animal, put to death animal behind intravenous injection Evans Blue (EB) dyestuff; get specimen; paraffin section, HE dyeing back light microscopic is observed [pressing Record R, Tuttle P; Tullius R; et al:Quantitative Assessment of Angiogensis in Response toImplants.Tissue Engineering, 2001,7 (5): the method described in 637] down.Utilize the blood capillary counting of optical microscope, can draw the microvessel density in the wound tissue institute's labelling in the above-mentioned section wound tissue.
According to the blood capillary count value of institute's labelling in the wound tissue, relatively add and do not add cytokine, and the wound surface different blood vessel degree that different adding method produced.The result is as follows:
● add to promote the active cell-less corium ground substance behind the somatomedin of angiogenesis, set up good blood circulation with the wound surface substrate at short notice after impelling cell-less corium ground substance to transplant, improved transplanting succeed rate, improved transplantation effect.
● the effect of group 1 is better than organizing 2 effect.
Embodiment 3
The preparation of active cell-less corium ground substance
Basically the step by embodiment 1 prepares active cell-less corium ground substance, and difference only is that the skin of selecting for use is the Corii Sus domestica after the depilation.
Embodiment 4
The preparation of active cell-less corium ground substance
Basically the step by embodiment 1 prepares active cell-less corium ground substance, and difference only is, is the VEGF replacement bFGF of 2500AU/ml with concentration.
Embodiment 5
The preparation of active cell-less corium ground substance
Basically the step by embodiment 1 prepares active cell-less corium ground substance, and difference only is, is that the mixed solution of 2500AU/ml bFGF is replaced former bFGF solution with concentration 2500AU/ml VEGF and concentration.
Embodiment 6
The animal experiment of active cell-less corium ground substance
Basically press the method for embodiment 2, the active cell-less corium ground substance for preparing among the embodiment 3-5 is carried out zoopery.The result shows that these active cell-less corium ground substances can be set up good blood circulation with the wound surface substrate at short notice, improves transplanting succeed rate, and improves transplantation effect.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (11)
1. graft is characterized in that it comprises:
(a) dermal matrix, cell quantity is pressed the stereometer of dermal matrix less than 5% in the described dermal matrix;
(b) cytokine of group under being selected from of effective dose: fibroblast growth factor, vascular endothelial cell growth factor, platelet derived growth factor, transforminggrowthfactor-or its mixture, wherein said effective dose are the 100-30000AU/ cubic centimetres.
2. graft as claimed in claim 1 is characterized in that described dermal matrix also contains the basement membrane complex.
3. graft as claimed in claim 1 is characterized in that, cell quantity is pressed the stereometer of dermal matrix less than 1% in the described dermal matrix.
4. graft as claimed in claim 1 is characterized in that, described cytokine is selected from: fibroblast growth factor, vascular endothelial cell growth factor or its mixture.
5. graft as claimed in claim 1 is characterized in that, described effective dose is the 200-10000AU/ cubic centimetre.
6. graft as claimed in claim 1 is characterized in that the thickness of described dermal matrix is 0.2-2mm, and described cytokine is a basic fibroblast growth factor.
7. graft as claimed in claim 1 is characterized in that, described dermal matrix is from the skin of following animal: pig, cattle, people, Mus.
8. a method for preparing graft is characterized in that, comprises step:
(a) provide a dermal matrix, the cell quantity in the described dermal matrix is pressed the stereometer of dermal matrix less than 5%;
(b) dermal matrix is contacted with the cytokine that is selected from down group, contain the described cytokine that concentration is the 100-30000AU/ cubic centimetre in the dermal matrix thereby make: fibroblast growth factor, vascular endothelial cell growth factor, platelet derived growth factor, transforminggrowthfactor-or its mixture.
9. method as claimed in claim 8 is characterized in that, in step (b), with the solution injection dermal matrix of needleless, high pressure filling gun with described cytokine; And the concentration of described solution is 240-24000AU/ml or 0.1-10ng/ml.
10. method as claimed in claim 8 is characterized in that, cell quantity is pressed the stereometer of dermal matrix less than 1% in the described dermal matrix.
11. a test kit is characterized in that, it contains the described graft of claim 1.
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