CN114259603A - Preparation method of embryonic chicken acellular matrix skin substitute - Google Patents
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Abstract
The invention belongs to the technical field of biomedical materials, and particularly relates to a preparation method of an embryonic chicken acellular matrix skin substitute, which comprises the steps of firstly treating the embryonic chicken skin by 0.25% of trypsin/0.02% of EDTA, removing cells of epidermis and dermis to obtain the embryonic chicken dermis, then treating the obtained embryonic chicken dermis by 0.1-0.5% of triton to destroy a residual cell structure to obtain an embryonic chicken dermis matrix, and finally treating the obtained embryonic chicken dermis matrix by Dnase I to remove residual cell fragments, so that the embryonic chicken acellular matrix skin substitute is obtained. The invention obtains relatively complete extracellular matrix, has simple preparation method, short time, high efficiency and light damage to the natural structure of the dermal extracellular matrix, and provides a better-quality dermal scaffold material for the treatment of large-area skin defects.
Description
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to a preparation method of an embryonic chicken acellular matrix skin substitute.
Background
The skin, the largest organ of the human body, is composed of the epidermis and the dermis, and is a natural barrier against the external environment and the invasion of pathogens. Skin defects can be caused by various factors, such as trauma, surgery, burns, and chronic ulcers. Among them, severe burning and trauma of the skin not only results in loss of normal function, but also endangers life. Therefore, the healing of the wound and the restoration of the normal structure and function of the skin appear to be critical for the healing of the patient.
When a large wound surface defect occurs, the original shape and function of the skin are difficult to restore by oneself. In this case, the wound defect is often repaired by means of skin flap and skin transplantation. Among them, autologous skin grafting is the most classical method for treating skin defects. However, patients with skin defects often have serious defects of skin donor areas, and have the problems of 'wound healing', non-ideal long-term effect, skin donor area infection, functional appearance defects and the like, so that new skin repair materials are needed to improve the current treatment situation of skin defects.
The development of tissue engineering has made various skin replacement products available for wound defects in nearly 50 million patients each year, but complete replacement of skin grafts has not been achieved. Temporary or permanent wound closure is currently achieved primarily by means of a scaffolding matrix, which protects the wound from infection, preventing further injury and loss of water. Currently, scaffolds for repairing skin defects mainly comprise synthetic polymers, biopolymers, degradable polymers or non-degradable polymers, etc., wherein the acellular biomaterials have a greater potential for repairing skin wound defects. Since decellularization is a tissue extracellular matrix separated from surrounding cells, artificial organs and tissues can be produced without adverse immune reactions after skin suppression. Since acellular biomaterials exhibit a relatively intact natural structure, good biocompatibility, has made them the focus of many studies. The acellular dermal matrix is a biomaterial formed by removing cell components and soluble proteins which cause host rejection reaction from allogenic or xenogenic skin and completely retaining natural morphological structures and composition components of a fiber network in the extracellular matrix, has the advantages of good mechanical property, low antigenicity and the like, has extremely similar components and structures with the microenvironment for normal cell growth, and is a scaffold material with the most application prospect at present. In addition to serving as a scaffold to support cell proliferation and migration during tissue development and regeneration, the extracellular matrix may provide a variety of biochemical and physical signals to facilitate the repair process.
Currently, there are four main methods for preparing natural dermal decellularized cells: (1) repeated freeze thawing; (2) chemical detergent methods; (3) a hypertonic salt method; (4) and biological enzyme digestion methods such as trypsin, neutral protease and nuclease. Meanwhile, in order to improve the cell removing effect, at present, more than two methods are generally adopted for removing cells sequentially or simultaneously, so that the manufacturing cost is reduced, and the cell removing speed and effect can be improved. However, the existing preparation method of the acellular dermal matrix still has the problems of complicated preparation, long time consumption, great damage to the natural structure of the dermal extracellular matrix and the like. Therefore, there is a need for a method of preparing decellularized dermis with a short cycle time, high efficiency, and low damage to the natural structure of the dermal extracellular matrix.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a preparation method of the embryonic chicken acellular matrix skin substitute, which has the advantages of short period, high efficiency and light damage to natural structures of dermal extracellular matrices.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the embryonic chicken acellular matrix skin substitute is prepared by treating embryonic chicken skin with 0.25% of trypsin/0.02% of EDTA, 0.1-0.5% of triton and Dnase I enzyme.
As a preferred embodiment of the present invention, the method for preparing the embryonic chicken acellular matrix skin substitute specifically comprises the following steps:
s1, 0.25% trypsin/0.02% EDTA treatment: soaking the skin of the embryonic chicken in 0.25% trypsin/0.02% EDTA, treating at 37 deg.C for 40-50min, taking out after the treatment, and cleaning to obtain the dermis of the embryonic chicken;
s2, 0.1-0.5% triton treatment: soaking the embryonic chicken dermis layer obtained in the step S1 in 0.1-0.5% triton for treatment for 45-90min, and taking out and cleaning after the treatment is finished to obtain an embryonic chicken dermis matrix;
s3, and treating with Dnase I enzyme: and (5) soaking the embryonic chicken dermal matrix obtained in the step (S2) in a DNase I enzyme solution, treating at 37 ℃ for 45-90min, taking out after the treatment is finished, and cleaning to obtain the embryonic chicken acellular matrix skin substitute.
Preferably, the method for obtaining the skin of the embryonic chicken comprises the following steps: and (3) hatching the fertilized eggs for nine days under the conditions that the temperature is 37 ℃ and the humidity is 60%, taking embryos, and cleaning to obtain the embryo chicken skins.
Preferably, in step S1, the processing time of 0.25% trypsin/0.02% EDTA is 40-50 min.
According to researches, the embryo chicken skin treated by 0.25% of trypsin/0.02% of EDTA is too long to damage the corium substrate, the skin is too short to achieve a good removal effect, the effect of removing cells cannot be achieved well when the embryo chicken is treated for 30min, and the corium substrate is damaged when the embryo chicken is treated for 60min and 90min, so that the optimal treatment time of 0.25% of trypsin/0.02% of EDTA is about 45 min.
Preferably, in step S2, the triton treatment concentration is 0.1% and the treatment time is 60 min.
It was found that when residual cells were destroyed by triton treatment of the embryonic chick dermal matrix, extracellular matrix components were destroyed by triton at too high a concentration or for too long a treatment time. The treatment concentration of 1% triton resulted in severe damage to the dermal matrix, while 0.05% triton did not achieve good results, and the treatment time of 120min also resulted in damage to extracellular matrix components, and less than 45min did not achieve good results. Therefore, in the triton treatment step, the optimum concentration of triton is 0.1%, and the optimum treatment time is 60 min.
Preferably, in step S3, the concentration of the DNase I enzyme solution is 7U/mL, and the treatment time of the DNase I enzyme is 55-65 min.
It has been found that when the cell debris is treated with nuclease, the mechanical properties of the material are greatly affected by too high enzyme concentration or too long treatment time. Since the optimum temperature of the DNase I enzyme activity is 37 ℃, when the enzyme treatment time is more than 90min, the material is broken, and when the enzyme treatment time is less than 45min, cell fragments cannot be removed well. Therefore, in the DNase I treatment step, the reaction time was about 60min at an enzyme concentration of 7U/mL and a reaction temperature of 37 ℃.
Preferably, the processes of steps S1 to S3 are all performed under rotation conditions, and the rotation speed is 60-80 r/min.
The invention also provides the embryonic chicken acellular matrix skin substitute prepared by the preparation method of the embryonic chicken acellular matrix skin substitute.
Compared with the prior art, the invention has the beneficial effects that:
the invention discloses a preparation method of an embryonic chicken acellular matrix skin substitute, which comprises the steps of firstly treating embryonic chicken skin by 0.25% of trypsin/0.02% of EDTA (ethylene diamine tetraacetic acid), removing epidermis and dermis cells to obtain an embryonic chicken dermis, then treating the obtained embryonic chicken dermis by 0.1-0.5% of triton to destroy a residual cell structure to obtain an embryonic chicken dermis matrix, and finally treating the obtained embryonic chicken dermis matrix by using Dnase I to remove residual cell fragments, so that the embryonic chicken acellular matrix skin substitute is obtained. The invention obtains relatively complete extracellular matrix, has simple preparation method, short time, high efficiency and light damage to the natural structure of the dermal extracellular matrix, and provides a better-quality dermal scaffold material for the treatment of large-area skin defects.
Drawings
FIG. 1 is a diagram of the morphology of embryonic chicken skin before and after decellularization;
FIG. 2 shows DAPI staining before and after acellular dermal layers of embryonic chickens;
FIG. 3 shows the DNA content changes before and after decellularization of embryonic chicken skin;
FIG. 4 shows the change in glycosaminoglycan (GAGs) content before and after the decellularization of the dermal layer of embryonic chickens;
FIG. 5 shows the Hydroxyproline (HYP) content change before and after decellularization of the acellular dermal layer.
Detailed Description
The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The experimental procedures in the following examples were carried out by conventional methods unless otherwise specified, and the test materials used in the following examples were commercially available by conventional methods unless otherwise specified.
Example 1 establishment of a method for preparing an acellular matrix skin substitute for embryonated chicken
1. Preparation method of embryonic chicken dermal matrix skin substitute
The preparation method comprises three steps of 0.25% tryptsin/0.02% EDTA treatment, triton treatment and DNase I enzyme treatment, namely, the skin of a fresh embryo chicken is treated by 0.25% tryptsin/0.02% EDTA to remove epidermis and dermis cells, then the residual cell structure is destroyed by triton treatment, and finally the residual cell debris is removed by DNase I treatment, so that the acellular matrix of the embryo chicken is obtained.
The preparation method comprises the following steps:
(1) 0.25% trypsin/0.02% EDTA treatment: removing epidermal and dermal layer cells
Putting the fertilized eggs into an incubator with the temperature of 37 ℃ and the humidity of 60% for incubation, taking embryos on the ninth day, putting the embryos into physiological saline for washing for 3 times, then taking the skins of the eggs by using sterilization forceps, putting the skins into 0.25% tryptsin/0.02% EDTA, reacting for 45min in a 37 ℃ water bath kettle at the rotating speed of 70r/min, putting the skins of the embryonated chicken into PBS for washing for 5 times after the reaction is stopped, and obtaining the corium of the embryonated chicken.
(2) triton treatment: destroying residual cellular structures
And (3) putting the embryonic chicken dermis layer obtained by the treatment in the step (1) into 0.1% triton, treating for 60min at a rotating speed of 70r/min, stopping reaction, and then putting into PBS (phosphate buffer solution) for washing for 3 times to obtain the embryonic chicken dermis matrix.
(3) Dnase i enzyme treatment: removing residual cell debris
And (3) putting the embryonic chicken dermal matrix obtained by the treatment in the step (2) into a culture dish with the diameter of 10cm, adding 7U/mL of DNase I enzyme solution, sealing the opening of the culture dish by using a sealing film, then putting the culture dish into a room with the temperature of 37 ℃ to react for 60min at the rotating speed of 75r/min, then rinsing the culture dish for 5 times by using PBS solution for 5min each time, further removing cell residues, taking out the dermal layer after the treatment is finished, and repeatedly rinsing the dermal layer by using deionized water to obtain the embryonic chicken dermal matrix skin substitute.
The morphology of the embryonic chick skin before and after decellularization is shown in fig. 1, and the effects of the embryonic chick dermal layer before and after decellularization are shown in fig. 2, and it can be seen that the embryonic chick dermal matrix is successfully prepared in this example.
2. Investigation of influence factors
(1) Optimal time for 0.25% trypsin/0.02% EDTA treatment
The treatment time of the embryo chicken skin with 0.25% trypsin/0.02% EDTA is too long, the corium substrate can be damaged, the good removal effect cannot be achieved after the treatment time is too short, and in order to explore the optimal treatment time of the 0.25% trypsin/0.02% EDTA, the treatment time is respectively set to be 30min, 45min,60min and 90min in the preparation process of the embryo chicken corium substrate skin substitute. As a result, it was found that the treatment for 30min failed to achieve a good cell removal effect, while the treatment time was 60min and 90min, which resulted in damage to the dermal matrix, the optimum time for the treatment of 0.25% trypsin/0.02% EDTA was about 45min (40-50 min).
(2) Optimal triton concentration and/or triton treatment time
When the residual cells are destroyed by treating the embryonic chicken dermal matrix with triton, the extracellular matrix components can be damaged by the triton with too high concentration or/and the triton for too long treatment time. To explore the optimal triton concentration or/and triton treatment time, triton concentrations were set to 0.05% triton, 0.1% triton, 0.5% triton, 1% triton, respectively, during the preparation of the embryonic chicken dermal matrix skin substitute; meanwhile, the treatment time was set to 45min,60min,90min, and 120min, respectively. It was found that when the treatment concentration was 1% triton, the dermal matrix was severely damaged, whereas 0.05% triton failed to achieve good results, and when the treatment time was 120min, the extracellular matrix components were also damaged, whereas less than 45min failed to achieve good treatment results. Thus, in the triton treatment step, the triton concentration is 0.1-0.5% and the treatment time is 45-90min, while the optimum concentration of triton is 0.1% and the optimum treatment time is 60 min.
(3) Optimal Dnase I treatment time
In the preparation process of the embryonic chicken dermal matrix skin substitute, certain cell fragments exist after 0.25% of trypsin/0.02% of EDTA and 0.1% of triton treatment, so nuclease treatment is required. During the DNase I treatment process, the mechanical properties of the material are greatly influenced by too high enzyme concentration or/and too long treatment time. In order to search for the optimal enzyme treatment time, in the preparation process of the embryonic chicken dermal matrix skin substitute, the optimal temperature of the DNase I enzyme activity is 37 ℃, the enzyme concentration is set to be 7U/mL, and the treatment time is respectively set to be 45min,60min,90min and 120 min. When the enzyme treatment time is more than 90min, the material is broken, and when the enzyme treatment time is less than 45min, cell fragments cannot be well removed. Therefore, in the step of treating with DNase I, when the enzyme concentration is 7U/mL and the reaction temperature is 37 ℃, the reaction time is 45-90min, and the optimum reaction time is about 60min (55-65 min).
3. Determination of the Properties of the embryonic Chicken dermal matrix
The DNA content of the embryonic chick acellular dermal matrix prepared in example 1 and the untreated fresh embryonic chick skin were measured, and as shown in fig. 3, it was found that the cells were well removed, and as shown in fig. 2, the DAPI staining also confirmed the result, and only a very small amount of nuclei were distributed in the embryonic chick acellular dermal matrix, confirming that the cells were successfully removed.
The embryonic chicken acellular dermal matrix prepared in example 1 and untreated fresh embryonic chicken skin were subjected to measurement of glycosaminoglycan (GAGs) content, and the results are shown in fig. 4, demonstrating that glycosaminoglycan in extracellular matrix components was well retained.
The embryonic chicken acellular matrix prepared in example 1 and untreated fresh embryonic chicken skin were subjected to measurement of hydroxyproline (a specific amino acid in collagen, which is an important index for measuring the metabolism of collagen tissues in the body) content, and the results are shown in fig. 5, which proves that the collagen content is well preserved after the acellular treatment.
The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.
Claims (8)
1. The preparation method of the embryonic chicken acellular matrix skin substitute is characterized in that the embryonic chicken acellular matrix skin substitute is prepared by treating embryonic chicken skin with 0.25% of trypsin/0.02% of EDTA (ethylene diamine tetraacetic acid), 0.1-0.5% of triton and DNase I.
2. The method for preparing the acellular matrix skin substitute for the embryonic chicken according to claim 1, which is characterized by comprising the following steps:
s1, 0.25% trypsin/0.02% EDTA treatment: soaking the skin of the embryonic chicken in 0.25% trypsin/0.02% EDTA, treating at 37 deg.C for 40-50min, taking out after the treatment, and cleaning to obtain the dermis of the embryonic chicken;
s2, 0.1-0.5% triton treatment: soaking the embryonic chicken dermis layer obtained in the step S1 in 0.1-0.5% triton for treatment for 45-90min, and taking out and cleaning after the treatment is finished to obtain an embryonic chicken dermis matrix;
s3, and treating with Dnase I enzyme: and (5) soaking the embryonic chicken dermal matrix obtained in the step (S2) in a DNase I enzyme solution, treating at 37 ℃ for 45-90min, taking out after the treatment is finished, and cleaning to obtain the embryonic chicken acellular matrix skin substitute.
3. The method for preparing the embryonic chick acellular matrix skin substitute according to claim 1 or 2, wherein the embryonic chick skin is obtained by a method comprising the following steps: and (3) hatching the fertilized eggs for nine days under the conditions that the temperature is 37 ℃ and the humidity is 60%, taking embryos, and cleaning to obtain the embryo chicken skins.
4. The method for preparing the embryonic chick acellular matrix skin substitute according to claim 2, wherein in the step S1, the treatment time of 0.25% trypsin/0.02% EDTA is 40-50 min.
5. The method of claim 2, wherein the treatment concentration of triton in step S2 is 0.1%, and the treatment time is 60 min.
6. The method of claim 2, wherein in step S3, the concentration of the DNase I enzyme solution is 7U/mL, and the treatment time of the DNase I enzyme is 55-65 min.
7. The method for preparing the acellular matrix skin substitute for embryonated chicken as claimed in claim 2, wherein the steps S1 to S3 are performed under a rotation condition, and the rotation speed is 60-80 r/min.
8. The embryonic chick acellular matrix skin substitute prepared by the method for preparing the embryonic chick acellular matrix skin substitute according to any one of claims 1 to 7.
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2021
- 2021-12-28 CN CN202111621623.7A patent/CN114259603A/en active Pending
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Application publication date: 20220401 |