CN100415307C - Heterologous or heterogenic decelled epidermis substitute used for human fibroblast-like cell modification - Google Patents
Heterologous or heterogenic decelled epidermis substitute used for human fibroblast-like cell modification Download PDFInfo
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- CN100415307C CN100415307C CNB2006100270449A CN200610027044A CN100415307C CN 100415307 C CN100415307 C CN 100415307C CN B2006100270449 A CNB2006100270449 A CN B2006100270449A CN 200610027044 A CN200610027044 A CN 200610027044A CN 100415307 C CN100415307 C CN 100415307C
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- dermal substitute
- human fibroblasts
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- acellular dermal
- allosome
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Abstract
The present invention relates to a heterologous or heterogenic cell extracted dermal substitute which is modified by human fibroblasts and is used as a dermal support or a culture carrier for epidermal cells. Cell culture technology is adopted to inoculate human fibroblasts pretreated by mitomycin C on the surface of a heterologous or heterogenic cell extracted dermal substitute in common clinic use at present, and after culture in vitro, the dermal substitute with a single layer or multiple layers of human fibroblasts. The fibroblasts excrete various cell factors, growth factors and extracellular matrices during the culture process, so that compared with the existing cell extracted dermal substitute, the cell extracted dermal substitute of the present invention has the advantages of high biological activity, favorable compatibility and high vascularization speed and is also favorable for the adhesion and the proliferation of the epidermal cells inoculated on the surface. The cell extracted dermal substitute can be used as a culture carrier for epidermal cells to construct composite skin.
Description
Technical field
The present invention relates to medical science wound repair technical field, particularly a kind of allosome or xenogenesis acellular dermal substitute of modifying as the human fibroblasts of dermis scaffold or epidermis cell cultivation carrier.
Background technology
At present, allosome or xenogenesis acellular dermal substitute be as a kind of dermis scaffold, and Preliminary Applications is in the reparation of degree of depth skin injury wound surface and cicatrix shaping etc.On the other hand, the acellular dermal substitute also can be used as epidermis cell and cultivates carrier, can disposable reparation holostrome skin injury wound surface after the external structure Composite Skin.Allosome or xenogenesis acellular dermal substitute need in preparation process through processing such as enzymic digestion and detergents, because when removal has antigenic cell composition, parts of fine extracellular matrix such as laminin, hyaluronic acid, IV Collagen Type VI, chondroitin sulfate etc. are residual few, and the shortage of these extracellular matrix compositions has reduced the bioaffinity of acellular dermal substitute, is unfavorable for adhesion, the propagation of cell.Therefore it is slower that allosome or xenogenesis acellular dermal substitute are transplanted back vascularization speed, easily cause being covered on the acellular dermal substitute from the necrosis of body skin graft, infection.And when cultivating carrier as epidermis cell, epidermal growth, propagation slowly, with dermal matrix adhere to weak, be easy to come off.
Summary of the invention
The invention provides that a kind of bioaffinity is good, vascularization speed is fast, be suitable for epidermis cell adheres to and the human fibroblasts of propagation is modified allosome or xenogenesis acellular dermal substitute.
The present invention modifies with human fibroblasts present used clinically allosome or xenogenesis acellular dermal substitute, that is inoculates, cultivates human fibroblasts on this dermal substitute.Fibroblast secretes the various kinds of cell factor in incubation, somatomedin and extracellular matrix composition, as interleukin-6, interleukin 8, transforming growth factor-beta, keratinocyte growth factor, acid fibroblast growth factor, basic fibroblast growth factor, laminin, hyaluronic acid, IV Collagen Type VI etc., make the acellular dermal substitute have higher biologic activity, improve affinity, being suitable for epidermis cell adheres to, growth and propagation, thus it is low to overcome acellular dermal substitute affinity commonly used, vascularization is slower, be unfavorable for that epidermis cell adheres to and the defective of propagation.
The allosome that inventor fibroblast is modified or the preparation method of xenogenesis acellular dermal substitute are:
1. the method that adopts conventional enzymic digestion, detergent to soak prepares allosome or xenogenesis acellular dermal substitute (sees Takami Y for details, Matsuda T, Yoshitake M, etal.Dispase/detergent treated dermal matrix as a dermalsubstitute.Burns, 1996,22 (3): 182-190), thickness is 0.3mm-0.6mm.
2. inoculation human fibroblasts:, in the cell culture fluid that contains 10 μ g/ml ametycins, hatch the back by 2 * 10 with the 2-9 human fibroblasts in generation
5Individual/cm
2-4 * 10
5Individual/cm
2Cell density is inoculated in allosome or xenogenesis acellular dermal substitute surface, places cell culture fluid to cultivate 1-2 week, and fibroblast forms monolayer or multiple confluent monolayer cells diaphragm adheres to acellular dermal substitute surface, is dermal substitute of the present invention.Cell culture fluid can be selected from culture fluid such as DMEM or 1640.
During use, if as dermis scaffold, then fibroblast faces down, and allosome or xenogenesis acellular dermal substitute that human fibroblasts is modified are implanted wound surface routinely, covers from the body skin graft about 2 weeks of postoperative, but wound repairing then; If cultivate carrier as epidermis cell, then inoculate the epidermis cell of certain density in the one side that contains human fibroblasts, behind In vitro culture, form the Composite Skin that contains cuticular cellulose routinely, transplantation is in holostrome skin injury wound surface.
Through experiment, acellular dermal substitute of the present invention is compared with allosome that does not contain human fibroblasts or xenogenesis acellular dermal substitute, can obviously improve the acellular dermal substitute bioaffinity, promote vascularization, be covered in its surface from body skin graft survival rate height; Dermal substitute of the present invention surface can strengthen the adhesion of the epidermis cell of inoculation, and because of human fibroblasts was handled through mitomycin C, its growth rate is slow, does not exist competition to suppress phenomenon to the growth that is inoculated in its surperficial epidermis cell, helps the external structure and the transplanting of Composite Skin.
The specific embodiment
The preparation of dermal substitute of the present invention if use xenogenesis acellular dermal substitute, then can be pressed 3-4 * 10 on its surface
5Individual/cm
2The cell density inoculation gets final product in In vitro culture 1-2 week through the pretreated human fibroblasts of mitomycin C, and containing monolayer or answering the fibroblastic xenogenesis acellular dermal substitute of layer of formation can place cell culture fluid standby.
If use allosome acellular dermal substitute, then can press 2-3 * 10 on its surface
5Individual/cm
2Cell density inoculation is through the pretreated human fibroblasts of mitomycin C, after In vitro culture 1-2 week, with form contain monolayer or the fibroblastic allosome acellular dermal substitute of multiple layer places cell culture fluid standby.
The allosome acellular dermal substitute that embodiment 1. preparation human fibroblasts are modified
1. prepare allosome acellular dermal substitute:
Enzymic digestion, detergent infusion method with routine prepare allosome acellular dermal substitute, and thickness is 0.4mm;
2. inoculation human fibroblasts:
The human fibroblasts that will be cultured to for the 3rd generation routinely adds cell culture fluid, and cell concentration is 2 * 10
6Individual/ml, add ametycin again, making its final concentration is 10 μ g/ml, hatches about 3 hours under 37 ℃, then with 2 * 10
5Individual/cm
2Cell density is inoculated in allosome acellular dermal substitute surface with it, places the DMEM culture fluid to cultivate 10 days, forms to contain the allosome acellular dermal substitute that multiple layer human fibroblasts modified.
The xenogenesis acellular dermal substitute that embodiment 2. preparation human fibroblasts are modified
1. prepare xenogenesis acellular dermal substitute:
The tomography Corii Sus domestica is prepared xenogenesis acellular dermal substitute with conventional enzymic digestion, detergent infusion method, and thickness is 0.3mm;
2. inoculation human fibroblasts:
The human fibroblasts that was cultured to for the 5th generation is added the DMEM culture fluid, and cell concentration is 2 * 10
6Individual/ml, add ametycin again, making its final concentration is 10 μ g/ml, hatches about 3 hours under 37 ℃, then with 4 * 10
5Individual/cm
2Cell density is inoculated in xenogenesis acellular dermal substitute surface with it, places the DMEM culture fluid to cultivate 7 days, forms and contains the xenogenesis acellular dermal substitute that the monolayer human fibroblasts is modified.
The method for preparing Composite Skin with acellular dermal substitute of the present invention is as follows:
Contain the one side inoculation epidermis cell of human fibroblasts at allosome of the present invention or xenogenesis acellular dermal substitute, epidermis cell is meant the patient from the body surface chrotoplast, also can be the cell mixing from body surface chrotoplast and allosome epidermis cell, and cell inoculation density is 2 * 10
5-4 * 10
5Individual/cm
2, in the DMEM culture fluid, cultivate 1-2 week routinely, can form the Composite Skin that contains monolayer or multiple layer epidermis cell.
Show through a large amount of animal (SD rat) experiments and part clinic trial, allosome that human fibroblasts is modified or xenogenesis acellular dermal substitute are with face down wound surface after implantation depth burn, chronic skin ulcer or the cicatrix excision of cell, cover then from body and open the sword pachydermia greatly, average survival rate reaches 95%, and the average survival rate that does not contain the acellular dermal substitute of human fibroblasts only is 78%.
Experiment is proof also, Composite Skin with acellular dermal substitute preparation of the present invention, formed monolayer or multiple layer epidermis cell and dermal substitute adhere to closely, be difficult for breaking away from, this Composite skin average survival rate to the wound surface reaches 63%, and the Composite Skin survival rate for preparing with the acellular dermal substitute that does not contain human fibroblasts only is 25%.
Claims (3)
1. the allosome modified of a human fibroblasts or the preparation method of xenogenesis acellular dermal substitute, step is as follows: the method that (1) adopts conventional enzymic digestion, detergent to soak prepares allosome or xenogenesis acellular dermal substitute; (2) inoculation human fibroblasts: the human fibroblasts in 2-9 generation is hatched the back by 2 * 10 in the cell culture fluid that contains 10 μ g/ml ametycins
5Individual/cm
2-4 * 10
5Individual/cm
2Cell density is inoculated in allosome or xenogenesis acellular dermal substitute surface, places cell culture fluid to cultivate and gets final product in 1-2 week.
2. allosome or the xenogenesis acellular dermal substitute modified of the human fibroblasts of the described method of claim 1 preparation.
3. the allosome of the described human fibroblasts modification of claim 2 or the application of xenogenesis acellular dermal substitute in the preparation Composite Skin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100270449A CN100415307C (en) | 2006-05-30 | 2006-05-30 | Heterologous or heterogenic decelled epidermis substitute used for human fibroblast-like cell modification |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100270449A CN100415307C (en) | 2006-05-30 | 2006-05-30 | Heterologous or heterogenic decelled epidermis substitute used for human fibroblast-like cell modification |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1868548A CN1868548A (en) | 2006-11-29 |
| CN100415307C true CN100415307C (en) | 2008-09-03 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2006100270449A Expired - Fee Related CN100415307C (en) | 2006-05-30 | 2006-05-30 | Heterologous or heterogenic decelled epidermis substitute used for human fibroblast-like cell modification |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101366976B (en) * | 2008-09-03 | 2012-08-29 | 陕西瑞盛生物科技有限公司 | Humanized heterogenous cell epimatrix material and preparation method thereof |
| CN101361989B (en) * | 2008-09-03 | 2012-09-19 | 陕西瑞盛生物科技有限公司 | Double membrane tissue patching material and preparation method thereof |
| CN101361990B (en) * | 2008-09-03 | 2012-09-19 | 陕西瑞盛生物科技有限公司 | Double layer artificial skin and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997004720A1 (en) * | 1995-07-28 | 1997-02-13 | Isolagen Technologies, Inc. | The use of autologous dermal fibroblasts for the repair of skin and soft tissue defects |
| CN1466999A (en) * | 2002-07-10 | 2004-01-14 | 上海第二医科大学附属瑞金医院 | Active cell-less corium ground substance |
-
2006
- 2006-05-30 CN CNB2006100270449A patent/CN100415307C/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997004720A1 (en) * | 1995-07-28 | 1997-02-13 | Isolagen Technologies, Inc. | The use of autologous dermal fibroblasts for the repair of skin and soft tissue defects |
| CN1466999A (en) * | 2002-07-10 | 2004-01-14 | 上海第二医科大学附属瑞金医院 | Active cell-less corium ground substance |
Non-Patent Citations (2)
| Title |
|---|
| 促进异种无细胞真皮血管化和体外构建复合皮移植的实验研究. 肖仕初.第二军医大学博士学位论文. 2001 |
| 促进异种无细胞真皮血管化和体外构建复合皮移植的实验研究. 肖仕初.第二军医大学博士学位论文. 2001 * |
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