CN106563173B

CN106563173B - Acellular biological dermis material and preparation method and application thereof

Info

Publication number: CN106563173B
Application number: CN201610890806.1A
Authority: CN
Inventors: 周鹤; 赵子建; 孙雪丽; 史啟; 潘璐琪
Original assignee: Zhejiang Huazhen Medical Equipment Co ltd
Current assignee: Zhejiang Hua Zhen Medical Equipment Co., Ltd.
Priority date: 2015-10-13
Filing date: 2016-10-12
Publication date: 2020-05-08
Anticipated expiration: 2036-10-12
Also published as: CN106563173A

Abstract

The invention discloses a preparation method of a decellularized biological dermis material, which comprises the steps of (1) treating pigskin with a trypsin solution containing EDTA, washing, soaking with a detergent solution, oscillating and washing, (2) treating the treated material with a neutral protease solution, washing, soaking with a detergent solution, oscillating and washing, (3) treating the material obtained in the steps (1) and (2) for 1-3 times, treating the treated material with a α -1, 3-galactosidase solution, and washing to obtain the decellularized biological dermis material.

Description

Acellular biological dermis material and preparation method and application thereof

Technical Field

The invention relates to a biological skin material and an implanted biological material in the fields of surgery, plastic surgery, hernia repair, abdominal cavity repair, pelvic repair and the like, in particular to an acellular dermal material and a preparation method and application thereof.

Background

The biological material has been widely applied in clinic in the world, but the research and development work in this aspect of China is generally in a lagging state at present, and medical devices made of the absorbable material and prepared by the biological material have great demands. However, the skin is the largest tissue organ of the human body and the outer barrier of the human body, and the integrity of the skin plays an important role for human beings. After the skin is damaged in a large area due to various reasons such as burns (wounds), serious dysfunction of the human body can be caused, and even the life is threatened. The most effective method for repairing skin defects at present is autologous skin transplantation, but autologous skin is seriously deficient due to large-area burns and the like, and the cutting of autologous skin is also a wound for patients. Although allogeneic skin and xenogeneic seed coat as temporary skin substitute can be used for treating skin defect wound, the same effect as autologous skin can not be achieved due to factors such as immune rejection, and the like, and the effect of covering and protecting the wound can be achieved. Therefore, it is more important to find safe and reliable xenogeneic seed coats which are not rejected by human bodies.

Acellular Dermal Matrix (ADM) is a new tissue substitute that has emerged in the last decade at home and abroad. The matrix is prepared by completely removing epidermal layer and cell components in skin by physical and chemical methods, only retaining extracellular matrix containing collagen net frame in dermis, and has the functions of wound surface covering, tissue defect filling, tissue regeneration guiding and scaffold and the like. ADM does not generally induce rejection because it is completely devoid of the cellular components and major immunological activities of type i and type ii cytocompatible antigens. However, ADM retains the three-dimensional structure of normal collagen and the extracellular matrix containing the collagen scaffold in the dermis, and can provide a good scaffold structure for the regeneration of tissue cells.

ADM was first applied to the repair of large-area burn scab wound surfaces and found to be effective in reducing scar formation. In the field of plastic reconstruction surgery, the application of the method is widely expanded to cleft palate repair, nasal deformity correction, repair of eyelid and conjunctival layer defects, lip augmentation, breast prosthesis wrapping and the like. In addition, ADM has been widely used in many fields such as oral cavity gingival tissue defect repair, tendon and joint capsule repair in orthopedics, abdominal surgery abdominal wall or pelvic floor defect repair, hernia repair, etc., and has a huge market demand.

The ADM product which is currently introduced domestically and takes the pig dermal tissue as the source is extremely similar to the human skin in the aspects of structure, physicochemical properties and functions, particularly the pig skin is extremely similar to the human skin in the aspects of structure, physicochemical properties and functions, the surface area of the pig body is large, the skin source is rich, the pig skin has good air permeability, the plasma extravasation can be prevented, the wound infection can be prevented, the ADM product is used for treating the skin of a patient with scald and burn as early as 1965, the pig skin can be laid with a matrix and provide nutrition for epidermal cells, the pig skin can be proliferated and promoted to heal, the ADM product can be used as a skin product for healing after being applied to the scald and burn for a short time in 1965, the pig skin is used for treating the skin of the patient with galactoside, the pig skin can be used for replacing the galactoside wound surface after being contacted with the galactoside, the pig skin is used for treating the skin of the burn and burn, and the pig skin has the effect of healing after being contacted with the pig skin for a plurality of times of wounds, namely, the pig skin wound healing, the pig skin is replaced by the galactoside wound surface of the pig skin, the pig skin is used for healing, the pig skin is used for healing for a plurality of the wound surface, the pig skin is used for the wound surface of the pig skin, the pig skin is used for healing of the wound surface of a plurality of the wound surface, the pig skin is used for the wound, the wound surface of the pig skin, the pig skin is used for the pig skin, the pig skin is used for the pig skin is used.

Disclosure of Invention

The technical problem to be solved by the invention is to provide an acellular biological dermal material which is safe, non-toxic, has deimmunity and can meet clinical requirements.

The technical problem to be solved by the invention is to provide a preparation method of the acellular biological dermal material.

The invention finally aims to solve the technical problem of providing the application of the acellular biological dermal material.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a preparation method of a decellularized biological dermis material comprises the following steps:

(1) treating pigskin with trypsin solution containing EDTA, washing, soaking in detergent solution, vibrating and washing;

(2) treating the material obtained after the treatment in the step (1) with a neutral protease solution, washing, soaking in a detergent solution, vibrating and washing;

(3) and (3) after the materials are treated for 1-3 times in the steps (1) and (2), treating the treated materials with α -1, 3-galactosidase solution, and washing to obtain the material.

In the steps (1) - (3), the washing is carried out by using 10mM Tris-HCl aqueous solution of 0.5-0.9 wt% sodium salt. The sodium salt is sodium chloride and/or sodium phosphate, and the pH value of the Tris-HCl aqueous solution is 6.0-7.9.

In the step (1), the trypsin solution added with EDTA is phosphate buffer solution added with 0.02 wt% -0.05 wt% of EDTA and 0.15 wt% -0.3 wt% of trypsin. The phosphate buffer solution is preferably a sodium phosphate buffer solution with the pH value of 6.0-7.9, and more preferably a sodium phosphate buffer solution with the pH value of 7.2.

The enzyme activity of the trypsin is more than or equal to 1000-2000BAEE units/mg.

The enzyme activity unit of trypsin is defined as: under the conditions that the concentration of a substrate N-benzoyl-L-arginine ethyl ester (BAEE) is 1mmol/L, the optical path is 1cm, the wavelength is 253nm, the temperature is 25 ℃, and the measurement volume is 3.2mL, the light absorption value increases by 0.001 per minute (delta A/min is 0.001) to be 1 BAEE enzyme activity unit.

In the step (1), the pigskin is soaked in a trypsin solution added with EDTA for 12 to 24 hours (preferably 24 hours) at the temperature of 30 to 37 ℃ (preferably 37 ℃).

In the step (2), the neutral protease comprises DispaseI and dispase (DispaseI).

The enzyme activity of the dispase (Dispasei product number D4818| CAS number 42613-33-2| SIGMA) is more than or equal to 10 unit/mg. Definition of enzyme activity: hydrolysis of casein at 37 ℃ for 1 minute at pH7.5 produced 1umole (181ug) of tyrosine as 1 enzyme activity unit expressed in u/g.

Enzyme activity of Dispase II>0.8U/mg (37 ℃ C., casein as substrate). Definition of enzyme activity: 1U is the amount of enzyme which releases 1umol of Folin positive amino acid or peptide corresponding to tyrosine 1 min under the detection conditions, corresponding to 181PU (protease units) or 600U Japanese units

In the step (2), the neutral protease solution is Tris-HCl aqueous solution which contains 500-700U/L neutral protease, has a pH value of 6.0-7.9 (preferably a pH value of 7.2) and contains 0.5-0.9 wt% of sodium salt. Wherein the sodium salt is sodium chloride and/or sodium phosphate.

In the step (2), the material obtained after the treatment in the step (1) is soaked in a neutral protease solution at 30-37 ℃ (preferably at 30 ℃) for 12-24 hours (preferably 12 hours).

In the step (1) or (2), the detergent solution is a phosphate buffer solution of 0.05-0.15 wt% of an ionic detergent, the ionic detergent is a mixture of sodium dodecyl sulfate and TritonX-200, the two can be mixed in any ratio, and the total concentration is required to be within the range. The phosphate buffer solution is preferably a pH 6.0-7.9 sodium phosphate buffer solution, and more preferably a pH7.2 sodium phosphate buffer solution.

In the steps (1) and (2), the material is soaked in a descaling agent solution and continuously vibrated, and the material after enzyme treatment is soaked in the descaling agent at room temperature for 18-24 hours (preferably 20 hours) and continuously vibrated; the shaking can be replaced by stirring.

In the step (3), the α -1, 3-galactosidase solution is a phosphate buffer solution containing α -1, 3-galactosidase in an amount of 10-20 μ g/mL, wherein the phosphate buffer solution is preferably a sodium phosphate buffer solution with a pH of 6.0-7.9, and more preferably a sodium phosphate buffer solution with a pH of 7.2.

The enzyme activity of α -1, 3-galactosidase is not less than 20units/mg, and the enzyme activity is defined as that 1 mu mole of p-nitro-phenyl-a-D-galactopyranoside is hydrolyzed in 1 minute at 25 ℃ and pH6.5 to be 1 enzyme activity unit.

In the step (3), the material obtained after the treatment is treated by α -1, 3-galactosidase solution, and the material obtained after the treatment is soaked in α -1, 3-galactosidase solution at 25 ℃ -37 ℃ (preferably 37 ℃) for 2-4 hours (preferably 2 hours).

The acellular biological dermal material prepared by the method is also within the protection scope of the invention.

The application of the acellular biological dermal material in the implant type biological tissue material is also within the protection scope of the invention. Wherein, the implanted biological tissue material includes but is not limited to the fields of surgery, plastic surgery, hernia repair, abdominal cavity repair, pelvic repair and the like.

The application of the acellular biological dermal material in preparing the material for repairing skin wounds is also within the protection scope of the invention.

The acellular biological dermal material provided by the invention is safe, nontoxic, cell-free, α -1, 3-galactoside-free and immunogenicity-free, and can be used in the fields of surgical repair, oral gingival tissue repair, plastic reconstruction surgery and the like.

Compared with the prior art, the acellular biological dermis material has the advantages of being safe and nontoxic, free of cells, capable of removing α -1, 3-galactoside and immunogenicity, capable of maintaining a natural collagen structure, good in toughness and strength, capable of being used as an implanted biological tissue material in the fields of surgery, plastic surgery, hernia repair, abdominal cavity repair, pelvic repair and the like, capable of being used for skin wound repair, simple in preparation method, easy to operate, low in cost and good in application prospect.

Drawings

FIG. 1 is a graph comparing treated and untreated hematoxylin-eosin staining; wherein A, B has not been decellularized; C. d, cell removal treatment; magnification: 200 times.

FIG. 2 is a comparison of treated and untreated α -1, 3-galactoside immunofluorescent staining, wherein A was not decellularized and B was decellularized at a magnification of 200.

FIG. 3 is a graph showing immunohistochemical staining of type I, type III, type IV, and type VII collagens after treatment.

FIG. 4 is a graph of hyaluronic acid immunohistochemical staining after treatment; wherein, the amplification factor of A is 40 times; b, magnification is 200 times;

fig. 5 is a tensile strength of the biological dermal material.

Fig. 6 shows the tension state of the mechanical detection of the biological dermis material.

FIG. 7 is the seeding profile (96-well plate) in example 8.

FIG. 8 shows the absorbance value at 450 wavelength, OD, after cytotoxicity detection.

FIG. 9 is a plot of the absorbance values of FIG. 8 and the corresponding sample combinations.

Detailed Description

The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.

Example 1: preparing the acellular biological dermal material.

Taking the whole skin of the lateral abdominal wall and back of a healthy adult pig, washing and disinfecting, cutting into appropriate sizes, washing with 10mM Tris-HCl containing 0.5 wt% sodium phosphate and pH7.2 aqueous solution, soaking in 10mM Tris-HCl buffer solution containing 0.5 wt% sodium phosphate and pH 7.4 at 37 ℃ for 24 hours, taking out the skin, washing with 10mM Tris-HCl containing 0.5 wt% sodium phosphate and pH7.2 aqueous solution containing 0.5 wt% sodium phosphate, washing with 0.1 wt% TritonX-200 ionic detergent and 0.03 wt% sodium lauryl sulfate and pH7.2 aqueous solution at room temperature for 20 hours, further washing with 10mM Tris-HCl containing 0.5 wt% sodium phosphate and pH7.2 aqueous solution containing 0.7 wt% sodium lauryl sulfate and pH7.2 aqueous solution containing 10mM Tris-3-5 wt% sodium phosphate, washing with neutral Tris-3-7.7.7 mM Tris-7 pH 7.8 aqueous solution containing 0.7 wt% sodium lauryl sulfate, washing with neutral sodium galactoside and pH 0.7.7-5 wt% sodium phosphate, washing with 10mM Tris-5% sodium lauryl sulfate and pH 7.8 aqueous solution containing 0.7.7.7.8 pH 7.7.8 pH 7.7-7% sodium phosphate, washing with neutral sodium sulfate, washing with neutral Tris-5 wt% sodium sulfate and pH 7.8 aqueous solution containing 0.8 pH 8 sodium galactoside and 0.7-5 wt% sodium phosphate, further washing with neutral sodium sulfate, washing with 0.7-10 mM Tris-5 wt% sodium phosphate, adding 10-7 pH 8 pH7 pH 8 pH 7.7-7 pH 8 sodium sulfate, and 0.7 pH7 pH 7.8 pH7 pH 7.7.7 pH7 pH 7.8 pH7 pH 7.8 pH 8 pH 7.8 pH7 pH 7.8 pH7 pH 8 pH.

Example 2: preparing the acellular biological dermal material.

Taking the whole skin of the flank and back of a healthy adult pig, washing and disinfecting the whole skin, cutting the whole skin into appropriate sizes, disinfecting and washing the whole skin with 10mM Tris-HCl containing 0.9 wt% of sodium chloride and pH 6.7 aqueous solution, then soaking the whole skin in 10mM sodium phosphate buffer solution containing 0.03 wt% of EDTA and 0.3 wt% of trypsin (product No. T4799. CAS No. 9002-07-7. SIGMA) and pH6.5 at 30 ℃ for 12 hours, taking out the skin, washing the whole skin with 10mM Tris-HCl containing 0.9 wt% of sodium chloride and pH 6.7 aqueous solution, shaking and soaking the whole skin with 0.03 wt% TritonX-200 of ionic detergent and 0.08 wt% sodium dodecyl sulfate and pH 6.7 sodium phosphate buffer solution at room temperature for 18 hours, further soaking the whole skin with 10mM Tris-HCl and pH 6.7. 10. mu. 10. mu. 10. mu. 3. 10. mu. mu.7. mu. mu.7 aqueous solution of sodium chloride with ionic sodium sulfate, 3. sodium sulfate, adding sodium chloride, adding sodium citrate, adding sodium chloride, adding ionic detergent.

Example 3: preparing the acellular biological dermal material.

Taking the whole skin of the lateral abdominal wall and the back of a healthy adult pig, washing and disinfecting the whole skin, cutting the skin into appropriate sizes, washing the skin with 10mM Tris-HCl containing 0.7 wt% of sodium chloride and with a pH of 7.8, then soaking the skin in sodium phosphate buffer solution containing 0.03 wt% of EDTA and 0.2 wt% of trypsin (product No. T4799. CAS No. 9002-07-7. SIGMA) and with a pH of 7.8 at 34 ℃ for 18 hours, taking out the skin, washing the skin with 10mM Tris-HCl containing 0.7 wt% of sodium chloride and with a pH of 7.8 aqueous solution, washing the skin with 0.03 wt% of ion detergent TritonX-200 and 0.03 wt% sodium dodecyl sulfate and with sodium phosphate buffer solution with a pH of 7.8 at room temperature for 24 hours, further soaking the skin with 10mM Tris-HCl and with a pH of 7.8. 7. 10. mu. TM. TMTris-HCl and with a pH of sodium chloride (10. 7. mu. 20. mu. 7. mu. 20. mu. 20. CAS. 20. 7. 20. mu. 7. 10. mu. 10. PH of sodium chloride, 7.8. PH of sodium chloride, 7. mu. PH 3-7. mu. 10.8. mu. PH of sodium chloride, and with sodium chloride, adding sodium chloride and adding sodium phosphate buffer solution of sodium chloride, adding sodium chloride, sodium sulfate, and adding sodium chloride, and adding sodium chloride, sodium phosphate buffer solution of sodium chloride, and adding sodium phosphate buffer solution of 0.7.7.8. 7.7.7.7.8 to 0.7.7.

Example 4:

an experiment of the decellularization effect was performed using the decellularized biological dermal material obtained in example 1.

The acellular effect of the acellular biological dermal material is identified by an HE staining method.

HE staining method:

the principle is as follows: the HE staining method is one of hematoxylin-eosin staining methods and staining methods commonly used in paraffin sectioning technology. Hematoxylin is basic, and mainly makes chromatin in the nucleus and ribosome in the cytoplasm bluish, and eosin is acidic, and mainly makes cytoplasm and extracellular matrix red.

The specific implementation steps are as follows:

1. fixing tissue, dehydrating, transparentizing, soaking in wax, embedding, and making into paraffin section

2. Paraffin sections were HE stained: after slicing and baking the paraffin, soaking in xylene I for 10min → soaking in xylene II for 10min → soaking in xylene III for 10min → soaking in absolute ethyl alcohol for 3min → soaking in 95% ethyl alcohol for 3min → soaking in 90% ethyl alcohol for 3min → soaking in 80% ethyl alcohol for 3min → tap water washing for 3 times of control → hematoxylin dyeing for 5min → tap water washing for 3-4 times (whether the dyeing solution is cleaned or not can be observed under the microscope, and not, the cleaning is continued), → 1% hydrochloric acid and alcohol differentiation for 3-5s (two times when shaken inside) can be observed under the microscope, and washing with tap water for 3 times → 45 degree warm water for 2min → washing with tap water for 3 times (the next dyeing can be observed under the microscope), → 1% eosin water solution for 3 times (when observed under the microscope), → 90% ethyl alcohol → 95% ethyl alcohol → 100% ethyl alcohol soaking (several times when shaken inside) → xylene (2) → ii (2) → min (when standing and stopping the tablet) The photograph was taken the next day on a dry air in a kitchen.

3. The photographing result is shown in FIG. 1. FIG. 1 shows that the cells were removed after the preparation method of example 1.

Example 5:

an immunogen removing effect test was performed using the acellular biological dermal material obtained in example 1.

The acellular effect of the acellular biological dermal material is identified by adopting an immunofluorescence staining method.

The specific implementation steps are as follows:

1. the embedded specimens were sliced with paraffin (5 microns) using a microtome and the wax slides were attached to polylysine-treated slides (commercially available, from Jiangsu Haiman, never dropped).

2. Baking at 60 deg.C for 2 hr

3. Xylene I was dewaxed for 30 minutes (which could be heated to 40-60 ℃ if complete dewaxing was to be ensured) and xylene II was dewaxed for 10 minutes.

4. Gradient alcohol rehydration: 100% alcohol for 3 minutes, 95% alcohol for 3 minutes, 85% alcohol for 3 minutes, 75% alcohol for 3 minutes, 50% alcohol for 3 minutes, distilled water for 3 minutes, and PBS immersion washing for 5 minutes.

5. And (3) performing antigen retrieval: placing the slices in 0.1mol/L citric acid solution with ph of 6.0, boiling with microwave oven 100 fire for three minutes to slightly boil, maintaining 50 fire for 7 minutes, stopping heating, and naturally cooling for 20-30 minutes.

6. The specimen was soaked in PBS for 3 minutes, the PBS outside the specimen was wiped off with filter paper, blocking serum was added dropwise, and blocking was carried out in a wet box at 37 ℃ for 30 minutes.

7. The blocking solution was wiped off with filter paper without washing. The fluorescence-anti-bacteria with proper concentration is dripped into a wet box and incubated overnight at 4 ℃ in the dark.

8. The primary antibody was washed off by 3 minutes × 3 washes with PBS, and the PBS outside the specimen was wiped off with filter paper.

9. Finally, the slides were mounted with glycerol and immediately observed and photographed under a fluorescent microscope.

10. The results after photographing are shown in FIG. 2.

FIG. 2 shows that α -1, 3-galactoside was not present in the tissue material after treatment with the preparation method of example 1.

Example 6:

the acellular biological dermal material obtained in example 1 retained the effects of collagen and hyaluronic acid components.

The acellular biological dermal material reserves collagen and hyaluronic acid components, wherein the I type collagen, the III type collagen and the hyaluronic acid are identified by an immunohistochemical SABC staining method, and the IV type collagen and the VII type collagen are identified by an immunohistochemical S-P staining method.

SABC method

1) Dewaxing and hydrating.

2) PBS was washed twice for 5 minutes each.

3) Preparing fresh 3% H2O2 with distilled water or PBS, sealing for 5-10 minutes at room temperature, and washing with distilled water for 3 times.

4) And (4) antigen retrieval.

5) PBS wash for 5 minutes.

6) Normal goat serum confining liquid is added dropwise, and the temperature is 20 minutes. And throwing off the redundant liquid.

7) Add I antibody dropwise at room temperature for 1 h or 4 ℃ overnight or 37 ℃ for 1 h (4 ℃ overnight followed by 37 ℃ rewarming for 45 min).

8) PBS was washed three times for 2 minutes each.

9) The biotinylated secondary antibody is dripped into the mixture for 20 minutes at the temperature of between 20 and 37 ℃.

10) PBC washes were 3 times for 2 minutes each.

11) Adding reagent SABC dropwise at 20-37 ℃ for 20 minutes.

12) PBS was washed 4 times for 5 minutes each.

13) DAB color development: and developing with a DAB developing kit or a self-prepared developer (mastering the developing degree under a mirror).

14) And (5) washing with distilled water. Hematoxylin counterstain for 2 minutes, and differentiation with hydrochloric acid and alcohol.

15) Dehydrating, transparentizing, sealing and microscopic examination.

SP method

1) Dewaxing and hydrating;

2) washing with PBS for 5 minutes for 2-3 times;

3) 3% H2O2 (80% methanol) was added dropwise to TMA, and the mixture was allowed to stand at room temperature for 10 minutes;

4) washing with PBS for 5 minutes for 2-3 times; 5) antigen retrieval;

6) washing with PBS for 5 minutes for 2-3 times;

7) normal goat serum confining liquid is added dropwise, and the temperature is 20 minutes. And throwing off the redundant liquid.

8) Mu.l of I-antibody was added dropwise and allowed to stand at room temperature for 1 hour or overnight at 4 ℃ or 1 hour at 37 ℃.

9) After 4 ℃ overnight, the temperature was rewarmed at 37 ℃ for 45 minutes.

10) PBS wash 3 times for 5 minutes each;

11) dripping 40-50 mu l of II antibody, and standing at room temperature or at 37 ℃ for 1 hour;

12) II antibody 0.05% tween-20 was added.

13) PBS wash 3 times for 5 minutes each;

14) DAB color development is carried out for 5-10 minutes, and the dyeing degree is grasped under a microscope;

15) rinsing with PBS or tap water for 10 minutes;

16) counterstaining with hematoxylin for 2 minutes, and differentiating with hydrochloric acid and alcohol;

17) washing with tap water for 10-15 minutes;

18) dehydrating, transparentizing, sealing and microscopic examination.

The photographing results are shown in fig. 3 and 4. FIGS. 3 and 4 illustrate that the collagen scaffold and hyaluronic acid components are retained after the above-mentioned preparation method.

Example 7:

the mechanical detection of the acellular biological dermal material obtained in example 1 maintains the toughness and strength of collagen.

The specific implementation is as follows

Width of skin sample detection: 3.4mm, thickness: 2.5mm, length: 36.1mm the stress-strain curve of the sample was measured under uniaxial tension at room temperature using a single column bench tester (5943, Instron, USA) with a sensor span of 1 kN. The displacement control is adopted in the experiment loading process, in the initial loading stage, in order to eliminate the influence of the defects of the sample, the sample is preloaded to 2N, then data recording is started, and the whole experiment process is carried out at the loading rate of 2 mm/min.

The analysis of the detection data is shown in FIG. 5. FIG. 5 illustrates that the tensile strength is above 10MPa in the mechanical detection, i.e., 85N/CM²As described above, fig. 6 shows a specific state of the stretching operation, and fig. 5 and 6 show that the biological dermal material has excellent toughness and strength. Example 8:

the acellular biological dermal material obtained in example 1 is used for detecting the cytotoxicity experiment, and the specific experiment is as follows:

1) the purpose and principle are as follows:

the purpose is as follows: detecting potential toxicological hazards of a product

The principle is as follows: CCK-8 agents are very low toxic to cells. It reacts with dehydrogenase in living cell continuously to deepen color and increase OD value. (Note: dehydrogenase is continuously produced in living cells). Therefore, the closer the OD value to the negative control group, the less toxicity, and the negative control group is responsible for the normal cell growth and the positive control group is toxic.

Reference: GB/T16886.5-2003 in vitro cytotoxicity assays; GB/T16886.12-2000 sample preparation and reference material

2) Reagent and apparatus

CCK-8 kit (Cat: CK04 Dojindo research institute of Endoconcha) ]; high-glucose DMEM cell culture medium; physiological saline

5ml centrifuge tube, 96-well plate, 24-well plate, culture dish

Filter paper, scalpel, ruler, beaker or erlenmeyer flask,

shaking table, incubator

3) The concrete operation steps

a. Sample preparation:

preparing a leaching solution: a0.6 cm by 0.5cm sample (surface area 1.26cm2) was taken; the samples were obtained by using the edge S of the decellularized biological dermal material obtained in example 1, and the middle S of the decellularized biological dermal material obtained in example 1, wild-type WT, respectively.

Soaking the samples in 1ml of cell culture medium, culturing at 37 ℃ and 100rpm for 24 h;

preparation of the control group: negative control NC: high density polyethylene (surface area 1.26cm2) leachate; positive control PC: 10% DMSO (100ul DMSO +900ul medium);

b. cell culture

Mouse fibroblasts (L929) were cultured in a 5% CO 237 ℃ incubator to near confluent state (plating 80%); preparing 10 × 104 cells/ml cell suspension, inoculating the suspension into a 96-well plate, adding 100ul of 104 cells into each well, and inoculating 7 × 5 cells in each well; discarding the culture medium when the cells grow to be in a nearly confluent state;

c. inoculation of leach liquor

100ul of negative control group leaching liquor and positive control, 100ul of 100% and 50% (50ul leaching liquor +50ul culture medium) of sample leaching liquor are respectively added into each well of a 96-well cell-grown well plate, 5 groups of leaching liquor are arranged in parallel, and 5% CO is added₂Continuously culturing for 24h in an incubator at 37 ℃; see FIG. 7;

4) cytotoxicity test

And (3) detecting cell proliferation toxicity by using a CCK-8 kit: the leaching solution was discarded, 100ul of new culture medium and 10ul of CCK-8 solution were added to each well, and OD450 was measured after incubation in the incubator for 2.5 h.

The detection result is shown in fig. 8, the data of fig. 8 and the corresponding samples are combined and arranged to be shown in fig. 9, and fig. 8 and 9 are detected OD values, and the detection principle and experimental data show that the acellular biological dermal material has very low toxicity to cells, so that the material is safe and nontoxic and can be used as an implanted biological material.

Claims

1. A preparation method of a decellularized biological dermal material is characterized by comprising the following steps:

(1) treating the pigskin after disinfection and washing with a trypsin solution added with EDTA, washing, soaking in a detergent solution, vibrating and washing;

(3) after the materials are treated for 1-3 times in the steps (1) and (2), the treated materials are treated by α -1, 3-galactosidase solution and washed to obtain the material;

wherein in the step (1) or (2), the detergent solution is 0.05-0.15 wt% of phosphate buffer solution of an ionic detergent, the pH value of the phosphate buffer solution is 6.0-7.9, and the ionic detergent is a mixture of sodium dodecyl sulfate and TritonX-200;

in the steps (1) - (3), the washing uses 0.5 wt% -0.9 wt% of Tris-HCl aqueous solution of sodium salt, and the pH value is 6.0-7.9;

in the step (1), the trypsin solution added with EDTA is a phosphate buffer solution added with 0.02 wt% -0.05 wt% of EDTA and 0.15 wt% -0.3 wt% of trypsin and having a pH value of 6.0-7.9;

in the step (2), the neutral protease solution is a Tris-HCl aqueous solution containing 500-700U/L neutral protease and 0.5-0.9 wt% of sodium salt, and the pH value is 6.0-7.9;

in the step (1), soaking pigskin for 12-24 hours at 30-37 ℃ by using a trypsin solution added with EDTA;

in the step (2), the material obtained after the treatment in the step (1) is soaked in a neutral protease solution at the temperature of 30-37 ℃ for 12-24 hours.

2. The method for preparing the acellular biological dermal material according to claim 1, wherein in the steps (1) and (2), the material is soaked in the detergent solution and continuously shaken, and the material after the enzyme treatment is soaked in the detergent at room temperature for 18-24 hours and continuously shaken or stirred.

3. The method for preparing the acellular biological dermal material according to claim 1, wherein in the step (3), the α -1, 3-galactosidase solution is phosphate buffer containing α -1, 3-galactosidase in an amount of 10-20 μ g/mL.

4. The method for preparing the acellular biological dermal material according to claim 1, wherein in the step (3), the processed material is processed by α -1, 3-galactosidase solution, and the processed material is soaked in α -1, 3-galactosidase solution at 25-37 ℃ for 2-4 hours.

5. An acellular biological dermal material produced by the method of any one of claims 1 to 4.

6. Use of the acellular biological dermal material of claim 5 for the preparation of an implant-type biological tissue material.

7. Use of the acellular biological dermal material of claim 5 for the preparation of a material for repairing skin wounds.