CN114177340A - Preparation and application of degradable placenta decellularized porous sponge matrix dressing - Google Patents

Preparation and application of degradable placenta decellularized porous sponge matrix dressing Download PDF

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CN114177340A
CN114177340A CN202111545807.XA CN202111545807A CN114177340A CN 114177340 A CN114177340 A CN 114177340A CN 202111545807 A CN202111545807 A CN 202111545807A CN 114177340 A CN114177340 A CN 114177340A
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placenta
porous sponge
freeze
freezing
acellular
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王铭祥
项宏
黄志军
王秀玉
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Hangzhou Beirong Biotechnology Co ltd
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Hangzhou Beirong Biotechnology Co ltd
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Priority to CN202111545807.XA priority Critical patent/CN114177340A/en
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Priority to CN202210411858.1A priority patent/CN115400255A/en
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    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/40—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing ingredients of undetermined constitution or reaction products thereof, e.g. plant or animal extracts
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Abstract

The invention relates to the field of biological materials, in particular to preparation and application of a degradable placenta decellularized porous sponge matrix dressing. Preparation and application of degradable placenta decellularized porous sponge matrix dressing comprise the following steps: (1) selecting materials from placenta tissues; (2) collecting and transporting placenta tissues; (3) inactivating the viruses of the placenta tissues; (4) removing the virus inactivation solution residue; (5) performing multiple circulating freeze thawing treatment; (6) a cell removal process; (7) removing the cell-free solution residue; (8) carrying out primary freeze drying; (9) freezing and ball milling; (10) digesting the obtained acellular freeze-dried powder; (11) adjusting the pH value of the obtained uniform digestion product, and pouring the uniform digestion product into freeze-drying molds of different specifications to obtain the acellular porous sponge; (12) sterilizing and inactivating viruses. The placenta acellular porous sponge scaffold prepared by the invention has the biodegradable characteristic, and can be used for rapid hemostasis, repair and regeneration of various acute and chronic wound surfaces.

Description

Preparation and application of degradable placenta decellularized porous sponge matrix dressing
Technical Field
The invention relates to the field of biological materials, in particular to preparation and application of a degradable placenta decellularized porous sponge matrix dressing.
Background
The placenta is an important organ for exchange of maternal and fetal substances, and is composed of amnion, chorion, and decidua basalis. Placenta is also an ideal biomaterial with good biocompatibility, promoting angiogenesis and inhibiting inflammation, wherein amnion has been used as a skin substitute for burns and ulcer lesions for over a hundred years. However, direct use or implantation may result in varying degrees of immune response, and decellularization techniques may minimize this adverse reaction. Compared with other tissue organs, the placenta is an important immunoregulation organ, and the acellular matrix contains more antioxidants, enzymes and abundant endogenous growth factors, such as Epidermal Growth Factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor-2 (FGF-2), Vascular Endothelial Growth Factor (VEGF), transforming growth factor beta (TGF-b) and the like, and can further promote tissue regeneration. In addition, compared with a single amniotic membrane substrate, the placenta substrate reserves abundant vascular systems, and the three-dimensional structure of the placenta substrate provides enough mechanical force to support the growth and development of new tissues, regulate and control the remodeling, degradation and regeneration of damaged tissues, and can be used for promoting wound healing, inhibiting fibrosis process and preventing postoperative scar contracture.
The extracellular matrix is recognized as the "soil" in which cells grow and includes collagen, elastin, proteoglycans and other nutrients. The extracellular matrix can regulate cell functions, promote the regeneration process of damaged tissues and play an important role in the tissue reconstruction process by simulating the natural microenvironment of cells. This is not comparable to any single collagen or hyaluronic acid. Due to the high complexity of the extracellular matrix, artificial fabrication is almost impossible to achieve, thus making use of the properties of decellularized tissue an emerging technology for tissue engineering and regenerative medicine. Although the porous collagen sponge material has been widely used for hemostasis and filling of wound surface in clinic, at present, no acellular porous sponge material which can be used in clinic is available. Common acellular materials are mostly in the form of patches, lack of porous structures and are difficult to fill, are formed by directly freeze-drying after tissue acellular, are difficult to ensure the efficacy consistency of products due to differences of tissue parts and batches, and lack of plasticity.
The decellularization and further preparation of placenta into porous sponge matrix dressings has been rarely studied. Chinese patent publication No. CN110975010A discloses a method for preparing placental extracellular matrix material, which comprises obtaining extracellular matrix gel by virus inactivation, decellularization and homogenization, but because of large particle size and uneven distribution, 6% of the gel can only pass through a 21G needle, and is barely injectable for use, the surface of the lyophilized sponge is rough, which affects the clinical effect, and the non-triple helical structure at the two ends of the collagen, i.e., telopeptide, cannot be effectively removed due to the lack of enzymatic hydrolysis process. Chinese patent publication No. CN104225667A discloses a method for preparing placenta extracellular matrix hydrogel, which can obtain homogeneous extracellular matrix hydrogel and porous sponge (pore diameter is 45-108 μ M), but the pepsin hydrochloric acid solution has an acid concentration of 0.1M, and is susceptible to excessive acidolysis due to its high acid concentration, so that the gel lyophilized powder cannot form gel at a concentration of 10mg/ml, and can stably form gel only at a concentration of about 30 mg/ml. Meanwhile, according to the animal-derived medical instrument registration technology review guide principle (2017 revision), the virus inactivation processes described in the above two publications have not been validated.
Disclosure of Invention
In order to achieve the purpose, the invention provides the following technical scheme: the preparation and application of the degradable placenta decellularized porous sponge matrix dressing specifically comprise the following steps:
(1) selecting materials for placenta tissues: fresh placenta delivered within 6 hours;
(2) collecting and transporting placenta tissues: collecting fresh placenta, cutting into small pieces, soaking in peroxyacetic acid solution for 0.5-4 hr, and storing placenta tissue at-8 deg.C for a long period; the placenta is in low temperature cold chain transportation (below 0 ℃);
(3) inactivating the viruses of the placenta tissues;
(4) removing virus inactivation solution residues: centrifuging the inactivated placenta, removing supernatant, adding purified water with the same amount for cleaning, centrifuging again, removing supernatant, and repeating for 2 times;
(5) the tissues are subjected to multiple circulating freeze-thaw treatments, wherein the freezing temperature is as follows: freezing at-80 deg.c to-50 deg.c for 6 hr or more; melting temperature: 23-28 ℃. Centrifuging and discarding the supernatant after each melting, and adding purified water with the same amount again for freezing;
(6) a cell removing process: treating the tissue treated in the step 5) with an enzyme solution, a non-ionic surfactant and an ionic surfactant solution for 60-90 min in sequence, and continuously stirring the mixture;
(7) removing the residue of the cell removing solution: cleaning the tissue treated in the step 6) with purified water for 15min, centrifuging and removing supernatant, and repeating for 4-6 times;
(8) carrying out primary freeze drying;
(9) freezing and ball milling: freezing and ball-milling the dried acellular placenta matrix obtained in the step 8) at the temperature of-20 to-50 ℃;
(10) digesting the acellular freeze-dried powder obtained in the step 9) by using a pepsin solution, wherein the digestion time is as follows: 24-96 hours;
(11) adjusting the pH value of the uniform digestion product obtained in the step 10) by using NaOH solution, pouring the uniform digestion product into freeze-drying molds with different shapes, pre-freezing for 6-8 hours at the temperature of minus 60-minus 48 ℃, performing gradient freezing and vacuum pumping, and increasing the temperature of each gradient by 5 ℃ to obtain the acellular porous sponge;
(12) sterilizing and inactivating viruses.
Preferably, the specific mode of the virus inactivation treatment of the placental tissues is as follows: the virus inactivation solution is prepared from peroxyacetic acid, absolute ethyl alcohol (more than or equal to 99.7 percent) and purified water according to a volume ratio of 3: 5: 92, mixing. 1kg of placenta tissue was treated with 19L of virus-inactivating solution for 0.5 to 1 hour.
Preferably, the first freeze-drying: placing the tissue treated in the step 7) into a freeze-drying tray, pre-freezing for 6-8 hours at-60 to-48 ℃, carrying out gradient freezing and vacuum pumping, and raising the temperature of each gradient by 5 ℃ to obtain the acellular freeze-drying sponge.
Preferably, the obtained dried acellular placenta matrix is subjected to freezing ball milling at the temperature of-20 ℃ to-50 ℃ to obtain a powder product with uniform particle size of less than 200 mu m.
Preferably, the ratio of the amount of pepsin in a 1mg/ml pepsin solution is 10mg (acellular tissue): the digestion is carried out in a proportion of 1ml (digestive juice), so that the product can be in an acidic (0.01M hydrochloric acid solution, and the pH value is 2.0-2.6) environment.
Preferably, the product after digestion and neutralization can be poured into molds of different thickness and shapes for freeze-drying.
According to the technical scheme, compared with the prior art, the virus inactivation process has short time and meets the requirements of technical review guidance principles of animal-derived medical instrument registration (revised 2017) after verification;
the decellularization process used by the invention removes cells and nucleic acid on one hand, and reserves extracellular matrix components and structures on the other hand;
through the freezing ball milling process, the ball milling process is kept at a low temperature, protein denaturation caused by overhigh temperature of products due to friction is avoided, and the proportion of ball milling balls can be randomly adjusted to adapt to different types of substances. More importantly, the single ball milling yield can exceed 50g, and large-scale batch production can be met;
through enzymatic digestion and freeze drying, the consistency of the sponge efficacy can be ensured, and the sponge has plasticity. On the other hand, the low immunogenicity of the product is ensured by cracking the collagen telopeptide;
the placenta acellular porous sponge scaffold prepared by the invention has the biodegradable characteristic, and can be used for rapid hemostasis, repair and regeneration of various acute and chronic wound surfaces. In addition, the implant can be applied to breast prosthesis operation, nose augmentation operation and the like to play a role in preventing adhesion and inhibiting fibrosis.
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 process flow diagram of the present invention.
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.
The embodiment of the invention discloses
Referring to the attached drawing 1, the preparation and application of the degradable placenta decellularized porous sponge matrix dressing disclosed by the invention specifically comprise the following steps:
(1) selecting materials for placenta tissues: fresh placenta delivered within 6 hours;
(2) collecting and transporting placenta tissues: collecting fresh placenta, cutting into small pieces, soaking in virus inactivating solution for 0.5-4 hr, and storing placenta tissue at-8 deg.C for a long period; the placenta is in low temperature cold chain transportation (below 0 ℃);
(3) inactivating the viruses of the placenta tissues;
(4) removing virus inactivation solution residues: centrifuging the inactivated placenta, removing supernatant, adding purified water with the same amount for cleaning, centrifuging again, removing supernatant, and repeating for 2 times;
(5) the tissues are subjected to multiple circulating freeze-thaw treatments, wherein the freezing temperature is as follows: freezing at-80 deg.c to-50 deg.c for 6 hr or more; melting temperature: 23-28 ℃. Centrifuging and discarding the supernatant after each melting, and adding purified water with the same amount again for freezing;
(6) a cell removing process: treating the tissue treated in the step 5) with an enzyme solution, a non-ionic surfactant and an ionic surfactant solution for 60-90 min in sequence, and continuously stirring the mixture;
(7) removing the residue of the cell removing solution: cleaning the tissue treated in the step 6) with purified water for 15min, centrifuging and removing supernatant, and repeating for 4-6 times;
(8) carrying out primary freeze drying;
(9) freezing and ball milling: freezing and ball-milling the dried acellular placenta matrix obtained in the step 8) at the temperature of-20 to-50 ℃;
(10) digesting the acellular freeze-dried powder obtained in the step 9) by using a pepsin solution, wherein the digestion time is as follows: 24-96 hours;
(11) adjusting the pH value of the uniform digestion product obtained in the step 10) by using NaOH solution, pouring the uniform digestion product into freeze-drying molds with different shapes, pre-freezing for 6-8 hours at the temperature of minus 60-minus 48 ℃, performing gradient freezing and vacuum pumping, and increasing the temperature of each gradient by 5 ℃ to obtain the acellular porous sponge;
(12) sterilizing and inactivating viruses.
Preferably, the specific mode of the virus inactivation treatment of the placental tissues is as follows: the virus inactivation solution is prepared from peroxyacetic acid, absolute ethyl alcohol (more than or equal to 99.7 percent) and purified water according to a volume ratio of 3: 5: 92, mixing. 1kg of placenta tissue was treated with 19L of virus-inactivating solution for 0.5 to 1 hour.
Preferably, the first freeze-drying: placing the tissue treated in the step 7) into a freeze-drying tray, pre-freezing for 6-8 hours at-60 to-48 ℃, carrying out gradient freezing and vacuum pumping, and raising the temperature of each gradient by 5 ℃ to obtain the acellular freeze-dried powder.
Preferably, the obtained dried acellular placenta matrix is subjected to freezing ball milling at the temperature of-20 to-50 ℃ to obtain a powder product with uniform particle size of less than 200 mu m, and the product with uniform particle size is beneficial to the next digestion.
Preferably, the ratio of the amount of pepsin in a 1mg/ml pepsin solution is 10mg (acellular tissue): the digestion is carried out according to the proportion of 1ml (digestive juice), so that the product is in an acidic (0.01M hydrochloric acid solution, and the pH is 2.0-2.6) environment, and the product is not easy to breed microorganisms.
Preferably, the product after digestion and neutralization can be poured into molds with different thicknesses and different shapes for freeze-drying so as to obtain porous sponge products with different shapes
According to the technical scheme, compared with the prior art, the virus inactivation process has short time and meets the requirements of technical review guidance principles of animal-derived medical instrument registration (revised 2017) after verification;
the decellularization process used by the invention removes cells and nucleic acid on one hand, and reserves extracellular matrix components and structures on the other hand;
through the freezing ball milling process, the ball milling process is kept at a low temperature, protein denaturation caused by overhigh temperature of products due to friction is avoided, and the proportion of ball milling balls can be randomly adjusted to adapt to different types of substances. More importantly, the single ball milling yield can exceed 50 g;
through enzymatic digestion and freeze drying, the consistency of the sponge efficacy can be ensured, and the sponge has plasticity. On the other hand, the low immunogenicity of the product is ensured by cracking the collagen telopeptide;
the placenta acellular porous sponge scaffold prepared by the invention has the biodegradable characteristic, and can be used for rapid hemostasis, repair and regeneration of various acute and chronic wound surfaces. In addition, the implant can be applied to breast prosthesis operation, nose augmentation operation and the like to play a role in preventing adhesion and inhibiting fibrosis.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other. The device disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.
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 (7)

1. The preparation and application of the degradable placenta acellular porous sponge matrix dressing are characterized by comprising the following steps:
(1) selecting materials for placenta tissues: fresh placenta delivered within 6 hours;
(2) collecting and transporting placenta tissues: collecting fresh placenta, cutting into small pieces, soaking in peroxyacetic acid solution for 0.5-4 hr, storing placenta tissue at-8 deg.C for a long time, and transporting placenta at low temperature (below 0 deg.C);
(3) inactivating the viruses of the placenta tissues;
(4) removing virus inactivation solution residues: centrifuging the inactivated placenta, removing supernatant, adding purified water with the same amount for cleaning, centrifuging again, removing supernatant, and repeating for 2 times;
(5) the tissues are subjected to multiple circulating freeze-thaw treatments, wherein the freezing temperature is as follows: freezing at-80 deg.c to-50 deg.c for 6 hr or more; melting temperature: 23-28 ℃. Centrifuging and discarding the supernatant after each melting, and adding purified water with the same amount again for freezing;
(6) a cell removing process: treating the tissue treated in the step 5) with an enzyme solution, a non-ionic surfactant and an ionic surfactant solution for 60-90 min in sequence, and continuously stirring the mixture;
(7) removing the residue of the cell removing solution: cleaning the tissue treated in the step 6) with purified water for 15min, centrifuging and removing supernatant, and repeating for 4-6 times;
(8) carrying out primary freeze drying;
(9) freezing and ball milling: freezing and ball-milling the dried acellular placenta matrix obtained in the step 8) at the temperature of-20 to-50 ℃;
(10) digesting the acellular freeze-dried powder obtained in the step 9) by using a pepsin solution, wherein the digestion time is as follows: 24-96 hours;
(11) adjusting the pH value of the uniform digestion product obtained in the step 10) by using a NaOH solution, pouring the uniform digestion product into a freeze-drying mold, pre-freezing for 6-8 hours at the temperature of minus 60-minus 48 ℃, performing gradient freezing and vacuum pumping, and raising the temperature of each gradient by 5 ℃ to obtain the acellular porous sponge;
(12) sterilizing and inactivating viruses.
2. The preparation and use of a degradable placental decellularized porous sponge matrix dressing according to claim 1, wherein transportation is used during transportation of placenta, temperature is recorded using a temperature recorder throughout the transportation, and product activity is maintained at low temperature.
3. The preparation and application of the degradable placental decellularized porous sponge matrix dressing according to claim 1, wherein the specific manner of the placental tissue virus inactivation treatment is as follows: the virus inactivation solution is prepared from peroxyacetic acid, absolute ethyl alcohol (more than or equal to 99.7 percent) and purified water according to a volume ratio of 3: 5: 92, mixing. 1kg of placenta tissue was treated with 19L of virus-inactivating solution for 0.5 to 2 hours.
4. The preparation and use of a degradable placental decellularized porous sponge matrix dressing according to claim 1, wherein said first freeze-drying: placing the tissue treated in the step 7) into a freeze-drying tray, pre-freezing for 6-8 hours at-60 to-48 ℃, carrying out gradient freezing and vacuum pumping, and raising the temperature of each gradient by 5 ℃ to obtain the acellular freeze-drying sponge.
5. The preparation and use of a degradable placental acellular porous sponge matrix dressing according to claim 1, characterized in that the obtained dried acellular placental matrix is subjected to cryo-ball milling at-20 ℃ to-50 ℃ to obtain a powder product with a uniform particle size of less than 200 μm, which is beneficial for the further digestion.
6. The preparation and use of a degradable placental decellularized porous sponge matrix dressing according to claim 1, wherein the matrix is prepared with 1mg/ml pepsin in a ratio of 10mg (decellularized tissue): the digestion is carried out according to the proportion of 1ml (digestive juice), so that the product is in an acidic (0.01M hydrochloric acid solution, and the pH is 2.0-2.6) environment, and the product is not easy to breed microorganisms.
7. The preparation and use of a degradable placental decellularized porous sponge matrix dressing according to claim 1, wherein the digested and neutralized product can be poured into molds of different thickness and different shape for lyophilization to obtain porous sponge products of different shape.
CN202111545807.XA 2021-12-16 2021-12-16 Preparation and application of degradable placenta decellularized porous sponge matrix dressing Pending CN114177340A (en)

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CN115006600A (en) * 2022-07-04 2022-09-06 杭州倍荣生物科技有限公司 Preparation method and application of temperature-sensitive cornea repair hydrogel with biological activity
CN115068661A (en) * 2022-06-21 2022-09-20 杭州倍荣生物科技有限公司 Calcium alginate composite porous biological matrix dressing, preparation method and application thereof
CN115887735A (en) * 2022-11-01 2023-04-04 湖南德普斯医疗科技有限公司 Preparation method of fungus stem acellular hemostatic sponge

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CN105999410B (en) * 2016-05-05 2020-04-07 广州昕生医学材料有限公司 Acellular tissue matrix composite material and preparation method thereof
CN109701078B (en) * 2019-02-22 2021-08-17 上海仁康科技有限公司 Biological sponge based on acellular dermal matrix and preparation method thereof
CN110975010B (en) * 2019-11-25 2022-02-15 银丰低温医学科技有限公司 Placenta tissue matrix material and preparation method thereof

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CN115068661A (en) * 2022-06-21 2022-09-20 杭州倍荣生物科技有限公司 Calcium alginate composite porous biological matrix dressing, preparation method and application thereof
CN115006600A (en) * 2022-07-04 2022-09-06 杭州倍荣生物科技有限公司 Preparation method and application of temperature-sensitive cornea repair hydrogel with biological activity
CN115887735A (en) * 2022-11-01 2023-04-04 湖南德普斯医疗科技有限公司 Preparation method of fungus stem acellular hemostatic sponge

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