CN101496915B - Heterogeneous dermis reticular layer stent without basement membrane and cell as well as preparation method thereof - Google Patents
Heterogeneous dermis reticular layer stent without basement membrane and cell as well as preparation method thereof Download PDFInfo
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- CN101496915B CN101496915B CN2009101030677A CN200910103067A CN101496915B CN 101496915 B CN101496915 B CN 101496915B CN 2009101030677 A CN2009101030677 A CN 2009101030677A CN 200910103067 A CN200910103067 A CN 200910103067A CN 101496915 B CN101496915 B CN 101496915B
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Abstract
The invention discloses an xenogenic dermal reticular layer scaffold with basilar membrane removal and no cells, which is taken from an dermal reticular layer of a mammal, and has a natural three-dimension structure with larger pores of the dermal reticular layer; the dermal scaffold removes a basilar membrane and has no cells; and the thickness of the dermal scaffold is between 0.2 and 0.8mm. The scaffold is prepared by performing sterile dermis getting, then removing subcutaneous tissue, epidermis, the basilar membrane and a dermal papillary layer, then orderly using distilled water to soak and using trypsinase and an EDTA.Na2 digestive juice to digest, using a phosphate buffer solution to wash after the persistent oscillation, then placing into a Triton X-100 solution for soaking and performing the persistent oscillation, and then using the phosphate buffer solution to wash. The scaffold completely removes the source of a main immunogenic substance in dermis, and reduces the immunogenicity; the scaffold has strong nutrition permeability, quick vascularization speed, high survival rate and good effect when used for the repair of skin wounds, the construction of tissue engineering skin, and tissue filling; and the scaffold has the advantages of rich dermis sources, simple preparation technology, short production period, and low cost.
Description
Technical field: the invention belongs to biomaterial for medical purpose, be specifically related to be used for a kind of xenogenesis reticular layer of corium support of skin wound reparation, organization engineering skin structure and tissue filling, and preparation method thereof.
Background technology: multiple clinically wound, disease can cause skin injury, large-area burns patient particularly, cut in early days crust, with the timely wound closure of dressing be repair at present skin the most effectively, the measure of most critical.Adopt patient's auto-skin grafting that scar hyperplasia and the Pigmented possibility that causes skin donor site arranged; Produce scar hyperplasia in various degree behind the wound healing, also there is the wretched insufficiency from body skin source in the serious burn patient.Therefore, seek the major subjects that a kind of ideal skin wound repair materials becomes repair in trauma.1913, Loewe confirmed that dermal tissue can strengthen the toughness of cutify, suppress granulation tissue growth and cicatrization, reduces wound surface contracture degree, and people just begin to pay close attention to the effect of dermal tissue in skin transplantation subsequently.Report such as Heck in 1985 is used allosome corium as the transplanting carrier from the body surface skin, and cograft is obtained better therapeutic effect to burn wound.Wainwright (1995) processes acellular dermal with human body skin, has researcher to transplant from the compound acellular dermal matrix of body surface chrotoplast diaphragm subsequently again and repairs the achieving success of holostrome skin injury.U.S. LifeCell company processes the acellular dermal support with this achievement commercialization with fresh people's corpse, called after AlloDerm, and the approval through U.S. FDA has got into biomaterial for medical purpose market.People's early starts such as domestic Chen Bi take off cell to allosome corium to be handled and clinical application research, and the allosome corium acellular dermal of existing similar Alloderm goes on the market at home at present.Because the source of people's allograft skin is extremely limited, far can not meet clinical needs, exploitation xenogenesis acellular dermal support becomes the effective way that addresses this problem.
Existing research proof, the main component and the skin immunization of skin basement membrane are closely related, and a lot of dermatosis (like systemic lupus erythematosus (sle), bullous dermatosis etc.) are related with the autoantibody of basement membrane zone; The basement membrane of xenogenesis acellular dermal and papillary layer immunoreation be (Srivastava 2001, and DeSagun 2001) the most by force; The somebody observes, and the antigenicity of pig dermis different levels also is not quite similar, and its antigenicity of reticular layer of corium that is positioned at than the position, deep is starkly lower than the papillary layer of corium than the superficial part position, shows that the reticular layer of corium of pig has immunologic inertia.Thereby basement membrane and papillary layer of corium are the immunogenic main sources that contains basement membrane xenogenesis acellular dermis.
It is ripe that the method that the allosome acellular dermal is made is tending towards gradually, and mainly contain balanced salt solution method, enzymic digestion-detergent method and height and ooze three kinds of salt-detergent method, but they have all kept basement membrane, and mainly from papillary layer of corium, like Alloderm.These methods are difficult to immunogenic substance is removed clean, still have immunological rejection in various degree after the transplanting.In the prior art, a December in 2003 disclosed CN1460527A on the 10th is arranged, it uses glutaraldehyde to reduce the immunogenicity of corium as chemical cross-linking agent, but has therefore changed the natural structure of corium fabric, makes corium fine and close, has influenced the implantation survival rate; Other has one in disclosed CN1343523A on April 10th, 2002; It has utilized the mode of laser boring to increase the hole and the permeability of support; Yet this mode gained hole is not the fiber natural structure, and laser has thermodenaturation to all collagen fiber in hole.The various xenogenesis acellular dermals of prior art still exist disadvantage: (1) cell eluting removes not thorough, and immunogenicity is stronger, the immunoreation of local long-term existence inflammation; (2) acellular dermal is fine and close, and to the poor permeability of nutrition, vascularization speed is slow, and survival rate is low after causing transplanting.
Summary of the invention
The objective of the invention is to the above-mentioned defective to the existence of prior art xenogenesis acellular dermal, intend a kind of acellular xenogenesis reticular layer of corium of basement membrane support and preparation method thereof that goes is provided, it is abundant to make it material source; The cell eluting is more thorough, and immunogenicity is lower; Keep natural fibre construction, hole is big, does not have chemistry and damage from laser again, and nutrition permeability is strong, vascularization speed is fast, survival rate is high.
Take following technical scheme to realize the object of the invention.
A kind of acellular xenogenesis reticular layer of corium of basement membrane support that goes is characterized in that:
1) it takes from the mammal reticular layer of corium, has the bigger natural three dimensional structure of reticular layer of corium hole;
2) dermis scaffold is removed basement membrane, and is acellular;
3) thickness of dermis scaffold is 0.2～0.8mm.
The above-mentioned acellular xenogenesis reticular layer of corium of the basement membrane support that goes, said mammal reticular layer of corium are the reticular layer of corium of animal of choosing any one kind of them in Cavia porcellus, bamboo rat, rat, coypu, rabbit, sheep, pig and the cattle.
Above-mentioned a kind of method for preparing of removing the acellular xenogenesis reticular layer of corium of basement membrane support is characterized in that operating according to the following steps:
1) obtains mammalian skin under the aseptic condition;
2) subcutaneous tissue of skin is removed by elder generation, removes epidermis, basement membrane and the papillary layer of corium of skin again;
3) with distilled water immersion 0.2 hour～3 hours;
4) using weight concentration is 0.18%～0.3% trypsin and 0.01%～0.035% EDTANa2 Digestive system, and 37 ℃ of following persistent oscillations 2 hours～8 hours, the reuse phosphate buffer was given a baby a bath on the third day after its birth time at least;
5) the good dermis scaffold of will going up step digestion places the TritonX-100 solution of weight concentration 0.3%～0.8% to soak and persistent oscillation 24 hours～48 hours, gives a baby a bath on the third day after its birth at least time with phosphate buffer;
That 6) will obtain goes the acellular xenogenesis reticular layer of corium of basement membrane support to be soaked in the 75% weight ethanol to preserve, or lyophilizing sealing back cobalt 60 illumination-based disinfections preserve, or preserves with oxirane disinfection after the lyophilizing.
That preserves removes the acellular xenogenesis reticular layer of corium of basement membrane support, cleans three times at least after soaking with normal saline and re-uses.
Remove the acellular xenogenesis reticular layer of corium of basement membrane support according to the inventive method obtained,, do not contain basement membrane, have the bigger natural three dimensional structure of hole, short texture, the big (see figure 2) of porosity owing to take from reticular layer of corium.This very helps nutrient permeation and blood vessel is grown into, and can set up effective blood circulation with the wound surface substrate at short notice, accelerates wound repair.
On method for preparing, fully removed the source of main immunogenic substance in the corium (basement membrane and papillary layer of corium), reduced immunogenicity; On the enzymic digestion of routine and detergent processing basis, improved operating procedure again; More increased the hypotonic processing of combination distillation water logging bubble; Make the eluting cell more thorough, reach acellular effect fully, further reduced immunogenicity (seeing Fig. 2, Fig. 3, Fig. 4).
Inoculate people's keratinocyte on the acellular xenogenesis reticular layer of corium of the basement membrane rack surface of the present invention going, cultivated 1 day, visible a plurality of cell adhesions are in the dermis scaffold surface, and the cell protuberance is not sprawled (see figure 5) as yet fully; Cultivated 5 days, cell is paved fully and is merged (see figure 6) in blocks gradually at rack surface; Cultivate after 15 days, it is transplanted to the nude mice back, postoperative formed more complete skin texture in 20 days, was dispersed in the support to be distributed with fibroblast and new vessels (see figure 7).
With the Corii Sus domestica skin be material source preparation remove the acellular xenogenesis reticular layer of corium of basement membrane support, with itself and the skin injury wound surface to rat back, can support microparticle skin (divergence ratio 2: 1) to survive and epithelization 2 all wound healings from body microparticle skin cograft; The wound surface shrinkage factor in 6 whens week only 35%～40%, and do not have the support matched group up to 70%～75%; Do not see that wound surface plays vesicle or the generation of festering 13 weeks, and do not see scar hyperplasia, gross examination of skeletal muscle is similar with surrounding skin, and touching with surrounding skin does not have obvious boundary (see figure 8).
The present invention removes the acellular xenogenesis reticular layer of corium of basement membrane support, and Pi Yuan is abundant, and preparation technology is simple, and fabrication cycle is short, and is cheap.
Description of drawings
Fig. 1 is the reticular layer of corium (H.E.100 *) that takes off before cell is handled
Annotate: do not see basement membrane, fibroblast is and is dispersed in distribution, and fibre structure is fine and close; It is thus clear that hair follicle and more sebaceous gland
Fig. 2 is for adopting the acellular xenogenesis reticular layer of corium of the no basement membrane support (H.E.100 *) of the inventive method preparation
Annotate: the inventive method can be removed the cell component in the reticular layer of corium fully, and the collagen bundle is complete, hole increases.
Fig. 3 is for lacking the acellular xenogenesis reticular layer of corium of the no basement membrane support (H.E.100 *) of hypotonic method preparation
Annotate: lack the prepared reticular layer of corium support of method of hypotonic processing it is thus clear that certain cell debris is residual
Fig. 4 does not have the acellular xenogenesis reticular layer of corium of basement membrane support (scanning electron microscope .300 *) for the present invention before the inoculating cell
Annotate: the surface is irregular uneven structure, and collagen fiber appear streak or " instant noodles appearance " structure, and many mesh that differ in size are arranged, and particularly do not see the residual component of cell.
Fig. 5 does not have 1 day (scanning electron microscope .3000 *) behind the acellular xenogenesis reticular layer of corium of the basement membrane support inoculation people keratinocyte for the present invention
Annotate: a plurality of cell adhesions are in the dermis scaffold surface, and the cell protuberance is not sprawled as yet fully.
Fig. 6 does not have 5 days (scanning electron microscope .1500 *) behind the acellular xenogenesis reticular layer of corium of the basement membrane support inoculation people keratinocyte for the present invention
Annotate: cultivate after five days, cell is paved fully and is merged in blocks gradually at rack surface.
Fig. 7 transplants people's keratinocyte compound no basement membrane acellular xenogenesis reticular layer of corium postoperative 20 days (H.E.40 *) for nude mice
Annotate: wound tissue section demonstration is real to have formed more complete skin texture, is dispersed in the support and is distributed with fibroblast and new vessels.
Fig. 8 does not have basement membrane acellular dermis lamina reticularis support cograft rat from body microparticle skin wound surface 6 weeks of postoperative for pig
The specific embodiment
Following apparatus system is equipped with instance and further specifies the present invention and go the acellular xenogenesis reticular layer of corium of basement membrane support method for preparing.
Embodiment 1:
As material source, it is following specifically to prepare process steps with rat:
1, aseptic rat back skin and the subcutaneous tissue of cutting;
2, placed 0.1% bromo geramine solution soaking 20 minutes;
3, give a baby a bath on the third day after its birth time with phosphate buffer (pH 7.2);
4, remove subcutaneous tissue with aseptic dermatome, obtain the holostrome skin of thickness 0.5mm;
5, the aseptic dermatome of reuse is removed epidermis, basement membrane and papillary layer of corium in the lump, obtains the reticular layer of corium (see figure 1) of thickness 0.3mm;
6, with the hypotonic reticular layer of corium that distilled water immersion obtained and persistent oscillation 130 times/minute 1 hour;
7, using weight concentration was the Digestive system of the EDTANa2 of 0.25% trypsin and 0.02%, 37 ℃ of following persistent oscillations 3 hours;
8, changing Digestive system is phosphate buffer, and persistent oscillation 10 minutes is given a baby a bath on the third day after its birth time repeatedly;
9, will digest good dermis scaffold and place weight concentration 0.5%TritonX-100 solution to soak, and persistent oscillation 130 times/minute 48 hours, give a baby a bath on the third day after its birth time with step 8;
10, again with dermis scaffold in freeze dryer-69 ℃ of following lyophilizing;
11, sealing back cobalt 60 illumination-based disinfections are preserved.
Embodiment 2:
As material source, it is following specifically to prepare process steps with rabbit:
1, aseptic rabbit back skin and the subcutaneous tissue of cutting;
2, placed 0.1% bromo geramine solution soaking 20 minutes;
3, give a baby a bath on the third day after its birth time with phosphate buffer (pH 7.2);
4, remove subcutaneous tissue with aseptic dermatome, obtain the holostrome skin of thickness 0.4cm;
5, the aseptic dermatome of reuse is removed epidermis, basement membrane and papillary layer of corium in the lump, obtains the reticular layer of corium of thickness 0.2mm;
6, with the hypotonic reticular layer of corium that distilled water immersion obtained and persistent oscillation 130 times/minute 1.5 hours;
7, use weight concentration be the Digestive system of EDTANa2 of 0.2% trypsin and 0.015% under 37 ℃, persistent oscillation 3 hours;
8, changing Digestive system is phosphate buffer, and persistent oscillation 10 minutes is given a baby a bath on the third day after its birth time repeatedly;
9, will digest good dermis scaffold and place weight concentration 0.3%TritonX-100 solution to soak, and persistent oscillation 130 times/minute 24 hours.Give a baby a bath on the third day after its birth time with step 8;
10, again with dermis scaffold in freeze dryer-69 ℃ of following lyophilizing;
11, sealing back cobalt 60 illumination-based disinfections are preserved.
Claims (3)
1. a method for preparing of removing the acellular xenogenesis reticular layer of corium of basement membrane support is characterized in that, according to the following steps operation:
1) aseptic rabbit back skin and the subcutaneous tissue of cutting;
2) placed 0.1% bromo geramine solution soaking 20 minutes;
3) use phosphate buffer, its pH is 7.2, gives a baby a bath on the third day after its birth time;
4) remove subcutaneous tissue with aseptic dermatome, obtain the holostrome skin of thickness 0.4cm;
5) the aseptic dermatome of reuse is removed epidermis, basement membrane and papillary layer of corium in the lump, obtains the reticular layer of corium of thickness 0.2mm;
6) with the hypotonic reticular layer of corium that distilled water immersion obtained and persistent oscillation 130 times/minute 1.5 hours;
7) use weight concentration be the Digestive system of EDTANa2 of 0.2% trypsin and 0.015% under 37 ℃, persistent oscillation 3 hours;
8) changing Digestive system is phosphate buffer, and persistent oscillation 10 minutes is given a baby a bath on the third day after its birth time repeatedly;
9) will digest good dermis scaffold and place weight concentration 0.3%TritonX-100 solution to soak, and persistent oscillation 130 times/minute 24 hours, give a baby a bath on the third day after its birth time with step 8);
10) again with dermis scaffold in freeze dryer-69 ℃ of following lyophilizing;
11) sealing back cobalt 60 illumination-based disinfections are preserved.
2. a method for preparing of removing the acellular xenogenesis reticular layer of corium of basement membrane support is characterized in that, according to the following steps operation:
1) aseptic rat back skin and the subcutaneous tissue of cutting;
2) placed 0.1% bromo geramine solution soaking 20 minutes;
3) use phosphate buffer, its pH is 7.2, gives a baby a bath on the third day after its birth time;
4) remove subcutaneous tissue with aseptic dermatome, obtain the holostrome skin of thickness 0.5mm;
5) the aseptic dermatome of reuse is removed epidermis, basement membrane and papillary layer of corium in the lump, obtains the reticular layer of corium of thickness 0.3mm;
6) with the hypotonic reticular layer of corium that distilled water immersion obtained and persistent oscillation 130 times/minute 1 hour;
7) using weight concentration was the Digestive system of the EDTANa2 of 0.25% trypsin and 0.02%, 37 ℃ of following persistent oscillations 3 hours;
8) changing Digestive system is phosphate buffer, and persistent oscillation 10 minutes is given a baby a bath on the third day after its birth time repeatedly;
9) will digest good dermis scaffold and place weight concentration 0.5%TritonX-100 solution to soak, and persistent oscillation 130 times/minute 48 hours, give a baby a bath on the third day after its birth time with step 8);
10) again with dermis scaffold in freeze dryer-69 ℃ of following lyophilizing;
11) sealing back cobalt 60 illumination-based disinfections are preserved.
One kind adopt that claim 1 or 2 said methods obtain remove the acellular xenogenesis reticular layer of corium of basement membrane support.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2009101030677A CN101496915B (en) | 2009-01-16 | 2009-01-16 | Heterogeneous dermis reticular layer stent without basement membrane and cell as well as preparation method thereof |
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| CN2009101030677A CN101496915B (en) | 2009-01-16 | 2009-01-16 | Heterogeneous dermis reticular layer stent without basement membrane and cell as well as preparation method thereof |
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| CN101496915A CN101496915A (en) | 2009-08-05 |
| CN101496915B true CN101496915B (en) | 2012-11-21 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102225218B (en) * | 2011-04-26 | 2014-07-09 | 中国人民解放军第三军医大学第一附属医院 | Method for preparing acellular dermal matrix by utilizing ultrasonic wave |
| CN103920195B (en) * | 2014-04-24 | 2015-05-13 | 刘邑卿 | Preparation method and application of clean fine-core sterile heterogeneous sigmoid membrane |
| CN105727367B (en) * | 2016-04-19 | 2021-03-26 | 王忠新 | Preparation method of heterogenous acellular dermal matrix |
| CN109321514B (en) * | 2018-06-11 | 2019-06-21 | 武汉奥翔生物科技有限公司 | Humanization skin and preparation method thereof |
| CN111437443A (en) * | 2020-04-20 | 2020-07-24 | 百澳瑞派（天津）生物科技有限公司 | Novel dermal matrix acellular method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1552467A (en) * | 2003-11-05 | 2004-12-08 | 中国人民解放军第三军医大学野战外科 | Abnormal decelled bone based material and its preparation |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1552467A (en) * | 2003-11-05 | 2004-12-08 | 中国人民解放军第三军医大学野战外科 | Abnormal decelled bone based material and its preparation |
Non-Patent Citations (1)
| Title |
|---|
| 杨燕妮等."异种无细胞真皮制备方法比较及在移植中的意义".《中国临床康复》.2004,第8卷(第23期),第4772页第2栏第2段-第4773页第2栏第1段. |
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