CN1257268C - Preparation and application of cell-eliminating pig corium substrate without cytotoxicity - Google Patents
Preparation and application of cell-eliminating pig corium substrate without cytotoxicity Download PDFInfo
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- CN1257268C CN1257268C CN 200410069394 CN200410069394A CN1257268C CN 1257268 C CN1257268 C CN 1257268C CN 200410069394 CN200410069394 CN 200410069394 CN 200410069394 A CN200410069394 A CN 200410069394A CN 1257268 C CN1257268 C CN 1257268C
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Abstract
In the present invention, the preparation of pig acellular dermis matrix comprises the steps that dermis matrix is placed in a solution containing sodium hydroxide of 0.5 Mol/L for continuous vibration, wherein the solution is prepared from tri-distilled water; tri-distilled water and phosphoric acid balanced saline are respectively used for rinse, and freeze drying, subpackaging and disinfection are then carried out. A tissue engineering compound skin preparing method by using pig acellular dermis matrix as an epidermal cell in vitro culture supporting frame comprises the steps that dermis matrix is coated with bovine collagen I dissolved in acetic acid, and the pH is adjusted to range from 7.0 to 7.4; the dermis matrix is soaked in a phosphoric acid balancing salt solution and a serum-free culture medium in sequence; after epidermal cells after inoculation and passage are taken out, the epidermal cells are placed in a CO2 incubator for serum-free culture. The application of pig acellular dermis matrix relates to the construction of tissue engineering compound skin through using the pig acellular dermis matrix as a supporting frame and the preparation of tissue engineering compound skin used as a human body skin substitute.
Description
Technical field
The invention belongs to medical science skin tissue engineering technical field, particularly relate to a kind of preparation method of cell-eliminating pig corium substrate without cytotoxicity; Relating to the cell-eliminating pig corium substrate without cytotoxicity is the method that support makes up the organizational project Composite Skin; Relate to cell-eliminating pig corium substrate without cytotoxicity and making up the application of organizational project Composite Skin and organizational project Composite Skin preparation as the skin quid pro quo.
Background technology
At present, cell-removed pig dermis matrix as the preparation method of human body skin quid pro quo mainly contain enzyme digestion, height oozes salt-detergent-treatment method and balance salt solustion mehtod three classes.Wherein, report is maximum both at home and abroad with enzyme digestion especially.It is under 4 ℃ or 37 ℃ of conditions, utilizes the effect of exogenous protease to remove epidermal structure, is aided with other method again, soaks and detergent-treatment etc. as tissue juice, reaches acellularization of dermal matrix; Height oozes salt-detergent-treatment method, is to utilize hypertonic salt solution to make anchor filament to separate with the hemidesmosome of epidermal basal cell, behind the complete removal epidermis, arrives acellularization of dermal matrix with detergent-treatment again.More than two kinds of methods, some chemical reagent of using in the preparation has extremely strong lysis effect as Tritonx-100, SDS etc., is difficult to thorough removing, causing with the dermal matrix is that support cell cultures success ratio is low, repeatable poor; The balanced salt solution method is that aseptic skin graft is positioned in 37 ℃ of phosphate buffered saline(PBS), and the skin endogenous protease is activated, thereby separates epidermis and corium, and methods such as molten with freezing repeatedly again, gammairradiation are removed whole intradermal survivaling cells.But this method, the one, the production technique more complicated, the treatment time is long, about about 2 weeks; The 2nd, easily pollute in the preparation process; The 3rd, antigenicity is removed not thorough.
Summary of the invention
One of task of the present invention is to provide a kind of chemical reagent to be eliminated easily, no antigen, and preparation cycle is short, the preparation method of the simple cell-eliminating pig corium substrate without cytotoxicity of manufacture craft;
Two of task of the present invention is to provide a kind of cell cultures success ratio height, the preparation method of the organizational project Composite Skin of favorable repeatability;
Three of task of the present invention is to provide a kind of cell-eliminating pig corium substrate without cytotoxicity making up the application of organizational project Composite Skin and organizational project Composite Skin preparation as the human body skin quid pro quo.
The preparation method of the said cell-eliminating pig corium substrate without cytotoxicity of the present invention, with anti-thick clean, the aseptic pigskin of 0.3-0.4mm that is whittled into, soaking the dermal matrix that obtains in the hypertonic saline through containing of tri-distilled water configuration, what place tri-distilled water configuration contains the 0.5Mol/L sodium hydroxide solution with sustained oscillation under the water-bath oscillator normal temperature 24 hours, with Ultrasonic Cleaners dermal matrix is washed 3 times with tri-distilled water gradation concussion at least again, at least concussion washing 10 minutes at every turn, with thorough removing wherein sodium hydroxide and the cell debris in the matrix.Use phosphoric acid balanced salt solution rinsing 5 times then, place in 95% the ethanol and soaked 30 minutes, use phosphoric acid balanced salt solution rinsing 3 times again after ,-80 ℃ of lyophilizes 4 hours, packing sterilization, cryopreservation.
The said cell-eliminating pig corium substrate without cytotoxicity of the present invention prepares the method for organizational project Composite Skin as epidermic cell vitro culture support, earlier dermal matrix is placed with behind the acetate dissolved I type bovine collagen solution endoperidium, adjustment PH is 7.0-7.4, again it is placed on earlier in phosphoric acid balanced salt solution and the serum free medium and soaked 48 hours, behind the epidermic cell of taking-up after its surface seeding goes down to posterity, placing temperature is 37 ℃, CO
2Concentration is 5% CO
2Serum-free culture in the incubator continues to cultivate 7-10 days after it being risen to the gas-liquid face in 48 hours.
In fact the application of the said cell-eliminating pig corium substrate without cytotoxicity of the present invention relates to cell-eliminating pig corium substrate without cytotoxicity and makes up organizational project Composite Skin and the preparation of the organizational project Composite Skin application as the human body skin quid pro quo as epidermic cell vitro culture support.
Go the application of cell pigskin dermal matrix preparation in order to understand the present invention better as the human body skin quid pro quo, with be that support carries out the organizational project Composite Skin surface of a wound transplantation effect that the epidermic cell vitro culture makes up with it, be that support carries out the comparative experiments that organizational project Composite Skin that cell cultures obtains and the simple epidermal sheet of cultivating repair the animal surface of a wound and proved with the cell-removed pig dermis matrix below by the present invention.
1. the preparation of epidermal sheet and organizational project Composite Skin
Get SD small rat holostrome skin, after being cell suspension with the DispaseII-tryptic digestion, the serum free medium that adopts Gibco company to provide carries out the epidermic cell vitro culture, and getting s-generation cell, to be inoculated in two groups of diameters respectively be in the culture dish of 60mm, and inoculum density is 0.25 * 10
6/ cm
2, the next day change liquid, promptly obtain the organizational project Composite Skin that the simple epidermal sheet of cultivating and the present invention contain multiple layer epidermis after 10 days.
2. experimental technique
Get 42 of healthy SD rats, male and female are not limit, body weight 205-250g.After every experimental mouse was pressed the capable intraperitoneal anesthesia of 50mg/Kg with 3% vetanarcol, the back unhairing cut away the full thick skin of the about 4cm of diameter, is divided into two groups at random.Use the organizational project Composite skin that contains multiple layer epidermis to cover for one group, another group is transplanted with the epidermal sheet of cultivating merely and is covered.All cover with petrolatum gauze and aseptic dressing after two groups of rat surface of a wound are transplanted, packing is fixing, and the single cage of animal is raised.2 weeks of postoperative, 4 weeks, 6 weeks are observed the wound healing rate, and shrinkage from mold dimensions is got graft tissue simultaneously and carried out histological observation.
3. experimental result
(1) gross examination of skeletal muscle
Compare organizational project Composite skin group, graft epithelization degree height with the epidermal sheet group, attach closely with surface of a wound substrate, smooth surface, wound healing is good, the wound healing rate is apparently higher than the epidermal sheet group, and shrinking percentage also obviously lowers (see Table 1, table 2).Show that the organizational project Composite Skin that the present invention makes up can repair the holostrome skin injury well, improve the wound healing quality.
Wound healing rate after table 1 organizational project Composite Skin and epidermal sheet are transplanted (%, X ± s)
| Group | The wound healing rate (%, X ± s) | ||
| Two weeks | All around | Six weeks | |
| Organizational project Composite Skin group epidermal sheet group | 78.65±2.69 * 68.23±2.23 | 85.06±2.13 * 74.17±1.96 | 92.66±2.87 * 82.19±2.35 |
*Organizational project Composite Skin group and epidermal sheet are formed the face healing rate relatively, P＜0.05, and significant difference is remarkable.
Surface of a wound shrinking percentage after table 2 organizational project Composite Skin and epidermal sheet are transplanted (%, X ± s)
| Group | Surface of a wound shrinking percentage (%, X ± s) | ||
| Two weeks | All around | Six weeks | |
| Organizational project Composite Skin group epidermal sheet group | 11.8±2.43 * 19.25±2.85 | 18.77±3.40 * 28.52±3.78 | 25.92±3.11 * 36.52±3.70 |
*Organizational project Composite Skin group and epidermal sheet are formed the face shrinkage ratio, P＜0.05, and significant difference is remarkable.
(2) histology is seen and is looked into
4 weeks of postoperative, organizational project Composite skin group, graft tissue is seen and is looked into visible more complete skin texture, the epidermal area multilayer structure is obvious, arrangement of collagen fibers is more regular in the skin corium, between a little inflammatory cell infiltration is arranged in the matter, visible abundant capillary structure is grown perpendicular to the surface of a wound, and more fibroblast proliferation is arranged; 6 weeks of postoperative, the visible complete skin texture of organizational project Composite Skin, epidermal area survive and layering near normal, arrangement of collagen fibers is regular in the skin corium, visible abundant capillary structure is grown perpendicular to the surface of a wound.The anti-Laminin dyeing of immunohistochemical methods shows that the organizational project Composite Skin is positive around basement membrane zone and corium blood vessel bundle, basement membrane zone dyeing is darker, and continuity is good, and the nipple structure of epidermis dermis joining region is obvious; The graft epidermal area of epidermal sheet group also has layering, but the disorder of intradermal arrangement of collagen fibers, inoblast is many.The anti-Laminin dyeing of immunohistochemical methods shows that also be positive, but it is shallow to dye, continuity is relatively poor around basement membrane zone and corium blood vessel bundle.
Above experimental result shows: use the organizational project Composite Skin that contains multiple layer epidermis that the present invention makes up, the wound healing rate is apparently higher than epidermal sheet, and the shrinking percentage of organizational project Composite Skin also obviously lowers than epidermal sheet after transplanting.Show that the present invention is that the organizational project Composite Skin that contains multiple layer epidermis that support makes up has good wound repair effect with the cell-removed pig dermis matrix.
Advantage of the present invention:
1. the dermal matrix of Huo Deing does not contain cellular constituent, no antigen;
2. in the preparation technology of dermal matrix and organizational project Composite Skin, take off cell preparation and dissolve I type bovine collagen bag by dermal matrix with the sodium hydroxide conduct respectively with acetate, the chemical reagent easy-clear, the dermal matrix nontoxicity that obtains, cell cultures success ratio height, favorable repeatability can obviously be improved the union of wounded skin quality after the transplanting;
3. preparation technology is simple, and fabrication cycle is short, and is cheap.
Embodiment
The preparation method of cell-eliminating pig corium substrate without cytotoxicity of the present invention cuts the thick pigskin skin graft into 0.3-0.4mm with counter, cleans to be placed in 75% alcohol and soaks three times, uses aseptic phosphoric acid balanced salt water washing 5 times again, and is clean, aseptic with the pigskin of guaranteeing to use.In order to remove the pigskin epidermis, present embodiment places the disinfectant after filtration of tri-distilled water configuration to contain 37 ℃ of immersions of 1Mol/L hypertonic saline 36 hours clean, aseptic pigskin.Then the dermal matrix that obtains is washed 3 times with tri-distilled water, be placed in the phosphoric acid balanced salt solution and soaked 2 hours.Be placed on after the taking-up in the sodium hydroxide solution of the tri-distilled water configuration that contains 0.5Mol/L, sustained oscillation is 24 hours under the usefulness water-bath oscillator normal temperature, and the concussion frequency is 100 times/minute.In order thoroughly to remove sodium hydroxide and the stroma cell fragment in the present invention's preparation, the preparation of dermal matrix of the present invention adopts Ultrasonic Cleaners to use tri-distilled water gradation concussion washing more than at least 30 minutes.The concussion washing pass that present embodiment provides is 4 times, each concussion washing 13 minutes.Afterwards, dermal matrix is placed in the phosphoric acid balanced salt solution rinsing 5 times, the alcohol immersion with 95% 30 minutes, use phosphoric acid balanced salt solution rinsing 3 times again after ,-80 ℃ of frozen dryings 4 hours, packing sterilization, cryopreservation.The dermal matrix of present embodiment after to packing adopts
60The Co illumination-based disinfection, 4 ℃ of refrigerators are preserved.
Cell-eliminating pig corium substrate without cytotoxicity of the present invention makes up the method for organizational project Composite Skin as epidermic cell vitro culture support, under aseptic condition, earlier with 0.5% acetate dissolving I type bovine collagen, adjustment PH is 7.0-7.4, then dermal matrix is placed by acetate dissolved I type bovine collagen solution, in order to wrap by the epidermis side of dermal matrix.To wrap again afterwards by after dermal matrix be placed on earlier in phosphoric acid balanced salt solution and the serum free medium, be 37 ℃, CO in temperature
2Concentration is to soak in 5% the incubator.After taking out in 48 hours, the epidermic cell after its epidermis side is inoculated going down to posterity of vitro culture.It is 37 ℃, CO that the dermal matrix that will inoculate epidermic cell then places temperature
2Concentration is to carry out the compound cultivation of serum-free in 5% the incubator, proceeds to cultivate after it being risen to the gas-liquid face in 48 hours, can obtain the organizational project Composite Skin in about about 10 days.
The I type bovine collagen that present embodiment provides is provided by Sigma company; Serum free medium is provided by Gibco company; The epidermic cell of dermal matrix epidermis side inoculation is the s-generation, and inoculum density is 0.25 * 10
6/ cm
2
Claims (4)
1. the preparation method of cell-eliminating pig corium substrate without cytotoxicity, it is characterized in that: aseptic pigskin is soaked the dermal matrix that obtains in the hypertonic saline of tri-distilled water configuration, the 0.5Mol/L sodium hydroxide solution that places tri-distilled water configuration was with sustained oscillation under the water-bath oscillator normal temperature 24 hours, with Ultrasonic Cleaners dermal matrix is washed 3 times with tri-distilled water gradation concussion at least again, at least concussion was washed 10 minutes at every turn, use phosphoric acid balanced salt solution rinsing 5 times then, place in 95% the ethanol and soaked 30 minutes, after using phosphoric acid balanced salt solution rinsing 3 times again,-80 ℃ of lyophilizes 4 hours, the packing sterilization, cryopreservation.
2. the method for preparing the organizational project Composite Skin with the cell-eliminating pig corium substrate without cytotoxicity of the described preparation method's preparation of claim 1 as epidermic cell vitro culture support, it is characterized in that: cell-eliminating pig corium substrate without cytotoxicity is placed with behind the acetate dissolved I type bovine collagen solution endoperidium, adjustment PH is 7.0-7.4, again it is placed on earlier in phosphoric acid balanced salt solution and the serum free medium and soaks, after taking out in 48 hours, epidermic cell after the going down to posterity of its epidermis side inoculation vitro culture, it is 37 ℃ that the cell-eliminating pig corium substrate without cytotoxicity that will inoculate epidermic cell then places temperature, CO
2Concentration is to carry out the compound cultivation of serum-free 48 hours in 5% the incubator, continues to cultivate 7-10 days after it is risen to the gas-liquid face.
3. cell-eliminating pig corium substrate without cytotoxicity according to claim 1 is used to make up the organizational project Composite Skin.
4. organizational project Composite Skin preparation according to claim 2 is as the application of human body skin quid pro quo.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410069394 CN1257268C (en) | 2004-07-22 | 2004-07-22 | Preparation and application of cell-eliminating pig corium substrate without cytotoxicity |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410069394 CN1257268C (en) | 2004-07-22 | 2004-07-22 | Preparation and application of cell-eliminating pig corium substrate without cytotoxicity |
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| CN1587390A CN1587390A (en) | 2005-03-02 |
| CN1257268C true CN1257268C (en) | 2006-05-24 |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100372511C (en) * | 2005-11-30 | 2008-03-05 | 烟台正海生物技术有限公司 | Acellular dermal matrix |
| CN100594041C (en) * | 2007-12-27 | 2010-03-17 | 南京医科大学附属南京儿童医院 | Preparation method for pig blood vessel acellular bracket by chemical and physical combination |
| CN102018991A (en) * | 2010-12-02 | 2011-04-20 | 南通大学 | Nano silver-porcine acellular dermal matrix (PADM) biological dressing and preparation method thereof |
| CN103341212B (en) * | 2013-06-28 | 2015-07-08 | 北京博辉瑞进生物科技有限公司 | Cleaning machine for preparing acellular tissue matrix material |
| CN112791224A (en) * | 2021-01-05 | 2021-05-14 | 海南大学 | Cross-linked composite collagen scaffold and preparation method thereof |
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2004
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