CN109701078B - Biological sponge based on acellular dermal matrix and preparation method thereof - Google Patents
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Abstract
The invention discloses a biological sponge based on an acellular dermal matrix and a preparation method thereof2+And fa-PEG. The biological sponge prepared by the invention is of a porous structure, the pores are communicated with each other, and the biological sponge has a three-dimensional structure similar to human skin tissue, so that organism tissue cells can grow rapidly; the product has good plastic formability, and can be prepared into different specifications according to requirements to meet the requirements; the biological sponge is an advanced tissue engineering scaffold material, contains rich growth factors, has good biocompatibility and is more favorable for supporting the growth of cells; the composite material can be used for wound repair, is an excellent tissue engineering skin scaffold material, and can be used for repairing skin defects and preparing tissue engineering skin.
Description
Technical Field
The invention relates to the technical field of biological materials, in particular to a biological sponge based on an acellular dermal matrix and a preparation method thereof.
Background
Skin defects are often caused by burns, wounds, diabetes, chronic ulcers, and the like. Pain, loss of physiological function, changes in appearance caused by intractable, chronic trauma and tissue defects severely affect the freedom of movement and quality of life of the patient and further cause psychological and social problems. Skin transplantation, which is the most effective treatment for tissue defects and skin complexion diseases, is limited in clinical use by the source and affects the treatment process. Tissue engineered skin is the most promising treatment. The current tissue engineering skin scaffold materials mainly comprise chemical synthesis materials such as: polylactic acid (PLA), poly-l-lactic acid (PLLA), and the like. The material has stable property, uniform structure, good mechanical property and poor biocompatibility. Acellular Dermal Matrix (ADM) is used as a natural extracellular matrix material, has good mechanical properties, histocompatibility and low immunogenicity, is a good matrix for tissue repair and reconstruction, is also a natural scaffold material commonly used in tissue engineering research, and has been widely used as a wound repair material for skin and soft tissue. At present, the methods for removing the dermal matrix mostly adopt an enzymatic hydrolysis method, a chemical reagent elution method and the like, and the methods all have the problems of great cytotoxicity and genetic toxicity of the obtained acellular dermal matrix caused by the fact that the acellular reagent cannot be completely removed.
After the cells of the natural material are removed, the natural material is processed by a certain cross-linking agent to have good mechanical properties, a good three-dimensional structure, good histocompatibility and low immunogenicity, and the method is a common method for tissue engineering research. However, these methods have certain problems: most of the existing cross-linking agents are reagents such as glutaraldehyde and the like, the reagents can cause pollution to the environment, and the cost is increased in subsequent treatment; most importantly, the residual reagent causes serious cytotoxicity, which greatly affects clinical use.
Therefore, it is an urgent need to solve the problems of the art to provide a biological sponge based on an acellular dermal matrix as a tissue engineering scaffold material for repairing skin defects and preparing tissue engineering skin.
Disclosure of Invention
In view of the above, the invention provides a biological sponge based on an acellular dermal matrix and a preparation method thereof, and the prepared biological sponge has good biocompatibility and good biological safety and is beneficial to supporting the growth of cells.
In order to achieve the purpose, the invention adopts the following technical scheme:
biological sponge based on acellular dermal matrix, which takes acellular dermal matrix as raw material and utilizes Ca2+And fa-PEG.
A preparation method of a biological sponge based on an acellular dermal matrix comprises the following specific steps:
(1) preparing an acellular dermal matrix:
firstly, taking stored human skin, and removing subcutaneous fat components;
cleaning and disinfecting the skin with subcutaneous fat components by using iodophor and alcohol, and repeatedly soaking and cleaning the skin by using a hypotonic solution;
thirdly, the skin cleaned in the second step is treated with 2.5mg/ml Dispase II solution overnight; discarding the Dispase II solution, removing epidermis, and then cleaning with normal saline to obtain dermis;
fourthly, Ca is contained in the genuine leather obtained in the third step2+Treating with hypertonic salt solution for 12-24 h; after the hypertonic saline solution is discarded, cleaning the solution by using normal saline;
fifthly, placing the dermis cleaned in the step IV into prepared cell removal liquid, and treating for 2-6 h in a dialysis device to obtain a cell removal dermis matrix;
sixthly, washing the obtained product for 3 times with normal saline, and washing the obtained product for 15min each time to obtain a human acellular dermal matrix;
(2) and (3) pepsin enzymolysis: adding 1% -5% pepsin solution into the acellular dermal matrix obtained in the step (1), and stirring and performing enzymolysis for 12-15h at room temperature;
(3) cleaning: filtering the acellular dermal matrix subjected to enzymolysis in the step (2) to obtain a precipitate, and washing the precipitate with normal saline;
(4) freeze-drying: freeze-drying the acellular dermal matrix sediment cleaned in the step (3) in a freeze dryer for 12-15 h;
(5) crushing: crushing the acellular dermal matrix precipitate obtained after freeze-drying in the step (4) for 3-5min by using an ultrafine crusher to obtain acellular dermal matrix powder;
(6) and (3) crosslinking:
adding distilled water into the acellular dermal matrix powder obtained in the step (5) to form a solution with the concentration of 0.01g/ml-0.5g/ml, then adding a cross-linking solution, and fully mixing; freezing the mixed solution at-20 deg.C for 12-24h, and lyophilizing to obtain biological sponge.
The crosslinking liquid isA certain amount of Ca2+And fa-PEG (Four-arm polyethylene glycol with succinimidyl glutamate Buffered gels) were fully dissolved in DPBS (Dulbecco's Phosphate Buffered Saline), Ca2+The concentration is 50-200. mu.M/L, and the concentration of fa-PEG is 0.01-0.5 g/ml.
Further, the step (1) of preparing the acellular dermal matrix: eliminating subcutaneous skin with fat component, washing with 2% iodophor, sterilizing, deiodinating with 75% alcohol, and soaking in sterile distilled water repeatedly.
Further, the step (1) of preparing the acellular dermal matrix: fourthly, 0.5 to 3mol/L Ca is contained in the genuine leather obtained in the third step2+Is treated by hypertonic salt solution for 12 to 24 hours, Ca2+The final concentration of (b) is 1-10 mM/L.
Further, the step (1) of preparing the acellular dermal matrix: step IV, the hypertonic salt solution contains 0.5 to 3mol/L of Ca2+Sodium chloride solution of (2), Ca2+The final concentration of (b) is 1-10 mM/L.
Further, the step (1) of preparing the acellular dermal matrix: the cell removing liquid comprises the following components: 0.1-0.5 wt% of pancreatin, 0.5-5mM EDTA, 0.1-0.5 wt% of TritonX-100, Na+0.1-0.5mol/L，Cl-0.1-0.5mol/L，Ca2+1-10mmol/L；Ca2+From Ca (NO)3)2，NO3 -2-20mmol/L。
Further, the step (1) of preparing the acellular dermal matrix: the cell removing liquid comprises the following components: 0.1-0.5 wt% of pancreatin, 0.5-5mM EDTA, 0.1-0.5 wt% of TritonX-100, Na+0.1-0.5mol/L，Cl-0.1-0.6mol/L，Ca2+1-10mmol/L；Ca2+From CaCl2。
According to the technical scheme, compared with the prior art, the invention discloses and provides the biological sponge based on the acellular dermal matrix and the preparation method thereof, the novel biological sponge is derived from allogeneic skin tissues, the structure of the novel biological sponge is consistent with that of human skin, and immune rejection reaction is very small after the acellular treatment;
the invention is beneficialUsing a novel cell-removing liquid containing Ca2+The components such as detergent, NaCl and the like, and the dermis structure is kept unchanged to the maximum extent while cells are removed; ca is also contained in the crosslinking liquid2+，Ca2+As an essential ion for cell culture, the acellular dermal matrix can not only stabilize the structure of the acellular dermal matrix under a proper concentration, but also has the functions of enhancing the mechanical, physicochemical, biocompatibility, hemostatic performance and the like of the material when the acellular dermal matrix is reconstructed into a 3D scaffold material;
the invention creatively introduces a dialysis device, removes harmful cell removal reagent while removing cells, and ensures low cytotoxicity, low genetic toxicity and high biocompatibility of the product;
the invention uses non-toxic novel cross-linking liquid to carry out cross-linking treatment, thus ensuring the biological safety of the obtained product; ca2+Polyethylene glycol is non-toxic, non-irritant and good in water solubility, is prepared into a 'cross-linking solution' according to a certain proportion, and is used for cross-linking and modifying an acellular dermal matrix to enable the acellular dermal matrix to have a loose three-dimensional space structure, improve the mechanical, physical and chemical properties, biocompatibility and the like, so that the acellular dermal matrix is applied to tissue engineering skin;
the biological sponge prepared by the invention is of a porous structure, the pores are communicated with each other, and the biological sponge has a three-dimensional structure similar to human skin tissue, so that organism tissue cells can grow rapidly; the product has good plastic formability, and can be prepared into different specifications (controllable thickness and size) according to requirements to meet the requirements;
the biological sponge is an advanced tissue engineering scaffold material, contains rich growth factors, has good biocompatibility and is more favorable for supporting the growth of cells; the composite material can be used for wound repair, is an excellent tissue engineering skin scaffold material, and can be used for repairing skin defects and preparing tissue engineering skin.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a schematic view of a dialysis device according to the present invention;
FIG. 2 is a photograph of an acellular dermal matrix obtained by the present invention;
FIG. 3 is an electron micrograph of the internal structure of the acellular dermal matrix of the present invention;
FIG. 4 is a graph showing the results of HE staining of normal skin tissue according to the present invention;
FIG. 5 is a graph showing the results of HE staining of the acellular dermal matrix according to the present invention;
FIG. 6 is a graph showing the staining of the acellular dermal matrix HE according to the present invention without the use of an acellular liquid and without the use of a dialysis device;
FIG. 7 is a scanning electron microscope image of a biological sponge prepared according to the present invention;
FIG. 8 is a drawing of a biological sponge of the present invention implanted in a skin wound to promote healing of the wound;
FIG. 9 is a graph showing the HE staining result of the biological sponge of the present invention transplanted on the skin wound to promote wound healing.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A preparation method of a human acellular dermal matrix comprises the following specific steps:
(1) preparing an acellular dermal matrix:
firstly, taking stored human skin, and removing subcutaneous fat components;
cleaning and disinfecting the skin with subcutaneous fat components by using iodophor and alcohol, and repeatedly soaking and cleaning the skin by using a hypotonic solution;
thirdly, the skin cleaned in the second step is treated with 2.5mg/ml Dispase II solution overnight; discarding the Dispase II solution, removing epidermis, and then cleaning with normal saline to obtain dermis;
fourthly, 2mol/L Ca content is used for the genuine leather obtained in the step three2+Is treated with sodium chloride solution for 18h, Ca2+The final concentration of (3) is 5 mM/L; after discarding the sodium chloride solution, cleaning with normal saline;
fifthly, placing the dermis cleaned in the step IV into prepared cell removal liquid, wherein the cell removal liquid comprises the following components: 0.1-0.5 wt% of pancreatin, 0.5-5mM EDTA, 0.1-0.5 wt% of TritonX-100, Na+0.1-0.5mol/L，Cl-0.1-0.5mol/L，Ca2+1-10mmol/L；Ca2+From Ca (NO)3)2，NO3 -2-20 mmol/L; treating in a dialysis device for 2-6 h to obtain acellular dermal matrix; the dialysis device is schematically shown in FIG. 1;
sixthly, washing with normal saline for 3 times, 15min each time, and obtaining the human acellular dermal matrix, as shown in figure 2;
a dialysis device: the allogeneic skin is placed in a dialysis bag, the acellular fluid enters from one end and flows out from the other end of the device, and harmful ions in the acellular fluid are dialyzed and removed while the acellular fluid is acellular, so that the cytotoxicity and the genetic toxicity of the acellular dermal matrix are reduced to the maximum extent while the acellular effect is ensured, and the biocompatibility is effectively improved.
Example 2
Internal structure of acellular dermal matrix
(1) Shearing the obtained acellular dermal matrix into a size of 1cm multiplied by 1cm, and sticking the acellular dermal matrix on a metal tray; hardening and embrittling the sectional acellular dermal matrix by adopting a liquid nitrogen quenching method, and then breaking; the section of the adhesive is upward and is adhered on a metal tray.
(2) After spraying gold in the sample preparation room, the sample is loaded on the machine, observed by a scanning electron microscope and photographed, and the result is shown in fig. 3.
As a result, no cell residue was observed in the tissue, and the collagen structure was intact, similar to that of normal skin.
Example 3 acellular dermal matrix HE staining
(1) Respectively placing normal skin tissues and acellular dermal matrixes in 4% paraformaldehyde fixing solution for fixing for 24 hours;
(2) dehydrating with alcohol, and making xylene transparent;
(3) dipping wax for 30min, embedding, and making a 5-micron thick sagittal paraffin section;
(4) dewaxing by gradient alcohol, dehydrating and HE dyeing;
(5) the results of HE staining of normal skin tissue by transparency, mounting and observation with a light microscope are shown in fig. 4, and the results of HE staining of acellular dermal matrix are shown in fig. 5.
As a result, no cell residue remained in the skin tissue.
Example 4
A preparation method of a human acellular dermal matrix comprises the following specific steps:
(1) taking stored human skin;
(2) removing subcutaneous fat components;
(3) removing the subcutaneous skin with fat components in the step (2), cleaning and disinfecting with 2% iodophor, deiodinating with 75% alcohol, and repeatedly soaking and cleaning the skin with sterile distilled water;
(4) treating the skin cleaned in the step (3) with 2.5mg/ml Dispase II solution overnight;
(5) discarding the Dispase II solution, removing epidermis, and then cleaning with normal saline to obtain dermis;
(6) mixing the corium obtained in step (5) with 2mol/L Ca2+Is treated with sodium chloride solution for 18h, Ca2+The final concentration of (3) is 5 mM/L;
(7) after discarding the sodium chloride solution, the mixture was washed with physiological saline for 15min 3 times to obtain an acellular dermal matrix of human origin, as shown in fig. 6.
Example 5
A preparation method of a biological sponge based on an acellular dermal matrix comprises the following specific steps:
(1) preparing an acellular dermal matrix;
(2) and (3) pepsin enzymolysis: adding a pepsin solution with the concentration of 3% into the acellular dermal matrix obtained in the step (1), and stirring and performing enzymolysis for 12 hours at room temperature;
(3) cleaning: filtering the acellular dermal matrix subjected to enzymolysis in the step (2) to obtain a precipitate, and washing the precipitate with normal saline;
(4) freeze-drying: freeze-drying the acellular dermal matrix sediment cleaned in the step (3) in a freeze dryer for 15 h;
(5) crushing: crushing the acellular dermal matrix precipitate obtained after freeze-drying in the step (4) for 5min by using an ultrafine crusher to obtain acellular dermal matrix powder;
(6) and (3) crosslinking:
adding distilled water into the acellular dermal matrix powder obtained in the step (5) to form a solution with the concentration of 0.3g/ml, then adding a crosslinking solution, and fully mixing; and (3) freezing the mixed solution at-20 ℃ for 18h, and freeze-drying to obtain the ADM biological sponge.
Example 6
Observing the biological sponge by an electron microscope:
1) taking materials, and placing biological sponge with diameter of 1cm and thickness of 1mm in electron microscope stationary liquid for fixing for 2 h;
2) dehydrating with ethanol, and dehydrating with 30%, 50%, 70%, 80%, and 90% ethanol concentration gradient for 1 time and 5 min/time, and dehydrating with 100% ethanol for 2 times and 5 min/time;
3) dissolving tert-butyl alcohol in warm water bath until ice crystals are dissolved, preparing 50%, 70%, 90% and 95% tert-butyl alcohol with anhydrous ethanol, and replacing ethanol from low to high according to concentration gradient for 1 time, 3 times and 10 min/time respectively for 100% tert-butyl alcohol;
4) spreading the sample in a culture dish, naturally airing, and storing in a sealed bag at 4 ℃ for later use;
5) placing the dried sample on a copper net, and spraying gold;
fixing the sample on the sample holder, observing by a scanning electron microscope and taking a picture, wherein the result is shown in figure 7; the results show that: the structure of the ADM biological sponge is regular, the pore size is uniform, and the ADM biological sponge is communicated with each other; a three-dimensional biological sponge with a delivery pore size structure is obtained.
Example 7
(1) Manufacturing a whole-layer skin defect nude mouse animal model with the diameter of 1cm by using a puncher, and simulating skin defect;
(2) after the full-layer skin defect is manufactured, a Control group (the wound surface of the group is only bound and is not covered with materials) and an ADM biological sponge group (the wound surface of the group is covered with ADM biological sponge and then is bound) are arranged.
The result is shown in fig. 8, 3d after the ADM biological sponge is transplanted, the wound surface of the Control group is still exposed, the exudation is obvious, and the wound surface area is increased compared with the original area; and ADM bio-sponge group: the wound surface is not exposed and does not exude. 7d after the ADM biological sponge is transplanted, the wound surface of the Control group is still exposed, a little exudation occurs, and the area of the wound surface is reduced to some extent; ADM biological sponge group: the wound surface is scabbed without exudation, and the area of the wound surface is obviously reduced compared with the initial area. 14d after the ADM biological sponge is transplanted, the wound area of the Control group is reduced, but the ADM biological sponge is not completely healed; ADM biological sponge group: the wound surface is basically healed. The result shows that the ADM biological sponge can promote the wound healing.
The result of the ADM biological sponge transplanted on the skin wound surface to promote the wound surface healing HE is shown in FIG. 9, and compared with the Control group, the ADM biological sponge wound surface healing effect is better.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (5)
1. A biological sponge based on acellular dermal matrix, characterized in that the biological sponge takes the acellular dermal matrix as raw material and utilizes Ca2+And fa-PEG for cross-linking;
the preparation method of the biological sponge based on the acellular dermal matrix comprises the following steps:
(1) preparing an acellular dermal matrix:
firstly, taking stored human skin, and removing subcutaneous fat components;
cleaning and disinfecting the skin with subcutaneous fat components by using iodophor and alcohol, and repeatedly soaking and cleaning the skin by using a hypotonic solution;
thirdly, the skin cleaned in the second step is treated with 2.5mg/mL Dispase II solution overnight; discarding the Dispase II solution, removing epidermis, and then cleaning with normal saline to obtain dermis;
fourthly, Ca is contained in the genuine leather obtained in the third step2+Treating with hypertonic salt solution for 12-24 h; after the hypertonic saline solution is discarded, cleaning the solution by using normal saline;
fifthly, placing the dermis cleaned in the step IV into prepared cell removal liquid, and treating for 2-6 h in a dialysis device to obtain a cell removal dermis matrix;
sixthly, washing the obtained product for 3 times with normal saline, and washing the obtained product for 15min each time to obtain a human acellular dermal matrix;
(2) and (3) pepsin enzymolysis: adding 1% -5% pepsin solution into the acellular dermal matrix obtained in the step (1), and stirring and performing enzymolysis for 12-15h at room temperature;
(3) cleaning: filtering the acellular dermal matrix subjected to enzymolysis in the step (2) to obtain a precipitate, and washing the precipitate with normal saline;
(4) freeze-drying: freeze-drying the acellular dermal matrix sediment cleaned in the step (3) in a freeze dryer for 12-15 h;
(5) crushing: crushing the acellular dermal matrix precipitate obtained after freeze-drying in the step (4) for 3-5min by using an ultrafine crusher to obtain acellular dermal matrix powder;
(6) and (3) crosslinking:
adding distilled water into the acellular dermal matrix powder obtained in the step (5) to form a solution with the concentration of 0.01g/mL-0.5g/mL, then adding a cross-linking solution, and fully mixing; freezing the mixed solution at-20 deg.C for 12-24h, and lyophilizing to obtain biological sponge;
the cell removing liquid comprises the following components: 0.1-0.5 wt% of pancreatin, 0.5-5mM EDTA, 0.1-0.5 wt% of TritonX-100, Na+ 0.1-0.5 mol/L，Cl- 0.1-0.5 mol/L，Ca2+ 1-10 mmol/L；Ca2+From Ca (NO)3)2，NO3 -2-20 mmol/L; or comprises the following components: 0.1-0.5 wt% of pancreatin, 0.5-5mM EDTA, 0.1-0.5 wt% of TritonX-100, Na+ 0.1-0.5 mol/L，Cl- 0.1-0.6 mol/L，Ca2+ 1-10 mmol/L；Ca2+From CaCl2；
The fa-PEG is Four-arm polyethylene glycol with succinimidyl glutamate terminated branches.
2. The acellular dermal matrix-based biological sponge according to claim 1, wherein the step (1) of preparing the acellular dermal matrix is: eliminating subcutaneous skin with fat component, washing with 2% iodophor, sterilizing, deiodinating with 75% alcohol, and soaking in sterile distilled water repeatedly.
3. The acellular dermal matrix-based biological sponge according to claim 1, wherein the step (1) of preparing the acellular dermal matrix is: fourthly, 0.5 to 3mol/L Ca is contained in the genuine leather obtained in the third step2+Is treated by hypertonic salt solution for 12 to 24 hours, Ca2+The final concentration of (b) is 1-10 mM/L.
4. The acellular dermal matrix-based biological sponge according to claim 3, wherein the step (1) of preparing the acellular dermal matrix is: step IV, the hypertonic salt solution contains 0.5 to 3mol/L of Ca2+Sodium chloride solution of (2), Ca2+The final concentration of (b) is 1-10 mM/L.
5. The acellular dermal matrix-based biological sponge according to claim 1, wherein the cross-linking solution contains Ca2+DPBS buffer with fa-PEG, Ca2+The concentration is 50-200 mu M/L,the fa-PEG concentration is 0.01g/mL to 0.5 g/mL.
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| CN111939321B (en) * | 2020-08-19 | 2021-07-30 | 西南大学 | Preparation method of pig acellular dermal matrix skin substitute |
| CN114177340A (en) * | 2021-12-16 | 2022-03-15 | 杭州倍荣生物科技有限公司 | Preparation and application of degradable placenta decellularized porous sponge matrix dressing |
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