CN112569346A - Hydrogel for promoting wound healing and preparation method thereof - Google Patents
Hydrogel for promoting wound healing and preparation method thereof Download PDFInfo
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- CN112569346A CN112569346A CN201910934665.2A CN201910934665A CN112569346A CN 112569346 A CN112569346 A CN 112569346A CN 201910934665 A CN201910934665 A CN 201910934665A CN 112569346 A CN112569346 A CN 112569346A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/38—Albumins
- A61K38/385—Serum albumin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
- A61K31/727—Heparin; Heparan
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1808—Epidermal growth factor [EGF] urogastrone
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
- A61K47/40—Cyclodextrins; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0014—Skin, i.e. galenical aspects of topical compositions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
Abstract
The invention belongs to the field of medicines, and relates to a hydrogel for promoting wound healing and a preparation method thereof. The hydrogel contains human epidermal growth factor hEGF, heparin sodium, human serum albumin, mannitol, beta-cyclodextrin, ethanol, propylene glycol, glycerol and other components. The hydrogel has a good promoting effect on wound healing and good stability.
Description
Technical Field
The invention belongs to the field of medicines, and relates to a hydrogel for promoting wound healing and a preparation method thereof.
Background
The skin is the largest organ of the human body and is also the first line of defense of the human body. In most cases, regeneration after skin injury is not difficult, and is regenerated through a series of overlapping processes, including initial immune response, tissue remodeling, cell proliferation and maturation. However, extensive damage deep into the dermis or hypodermis, such as deep II or III degree burns, can lead to difficult skin regeneration.
The healing process of the wound surface is a complex process which involves the processes of re-epithelialization of a skin defect part, proliferation of granulation tissue, inflammatory reaction, vascular proliferation, remodeling of the wound surface and the like, wherein any one of the processes has problems and can cause wound surface healing disorder. At present, the method for repairing the wound surface is mainly an operation, namely, an open wound is changed into a closed wound through operation suture, so that the healing of the wound surface is accelerated, but scars are left after the operation, the wound has overlarge tension and is subjected to recrudescence or functional disorder and other complications after healing. For some complex wound surfaces, such as radioactive wound surfaces, diabetic foot surfaces, wound surfaces after long-term hormone use and the like, the wound surfaces often have local blood circulation disorder, the bioactivity of fibroblasts is reduced, the differentiation and paracrine effects of mesenchymal stem cells are inhibited and the like, so that the regeneration of the epidermis of the wound surface is slow, the inflammatory reaction is reduced, the anti-infection capacity of the wound surface is reduced, and even if the wound surface is small, the wound surface is closed by adopting a skin grafting or direct suturing method and is not easy to heal.
In recent years, products such as gel, spray or dressing of recombinant bovine or human fibroblast growth factor (b-FGF), recombinant human EGF, recombinant human PDGF and other cytokines are on the market successively and are applied to clinic, and clinical test results of multi-center large samples show that the cytokine intervention treatment is adopted for superficial II-III degree burn wounds, lower limb varicose ulcers, diabetic feet, radiation ulcers, bedsores and other wounds singly or in combination, so that the wound healing can be accelerated, and the healing quality of the wounds can be obviously improved. There are still a number of disadvantages: these growth factors act singly; after acting on the wound surface, the protein kinase in the wound surface is degraded quickly and can not effectively and continuously act on specific target cells, so that the bioavailability of the protein kinase is reduced, and a satisfactory clinical effect cannot be obtained.
Disclosure of Invention
The invention aims to provide a hydrogel for effectively promoting wound healing.
It is another object of the present invention to provide a stable wound healing promoting hydrogel.
It is another object of the present invention to provide a long-acting wound-healing promoting hydrogel.
Another object of the present invention is to provide a hydrogel having high bioavailability for promoting wound healing.
The above object of the present invention is achieved by the following technical solutions:
the invention provides a hydrogel for promoting wound healing, which comprises the following components:
the pH of the hydrogel is 6-8.
In a preferred embodiment of the present invention, the hydrogel comprises the following components:
the hydrogel has a pH of 7-8.
In a preferred embodiment of the present invention,
the hydrogel comprises the following components:
as a preferred embodiment of the present invention, the hydrogel further comprises any one of the following components:
0 to 5 percent of preservative
0 to 5 percent of pH regulator
Chelating agent 1-10 uM.
As a preferred embodiment of the present invention, the hydrogel simultaneously contains the following components:
0 to 0.5 percent of preservative
0 to 0.05 percent of pH regulator
Chelating agent 1-10 uM.
As alternative embodiments, the preservative is not particularly limited, and in the prior art, the preservative used for the gel in the medical field may be any, including but not limited to: one or more of parabens, benzoic acid, sodium benzoate, benzalkonium chloride, benzalkonium bromide, chlorobutanol and sorbitol.
As an exemplary embodiment of the present invention, the preservative is selected from methyl paraben and propyl paraben.
As alternative embodiments, the pH adjusting agent is not particularly limited, and in the prior art, pH adjusting agents used in the medical field may be any, including but not limited to: one or more of sodium hydroxide, hydrochloric acid, phosphate, triethanolamine, sodium bicarbonate, citric acid, citrate, boric acid and borate.
As an exemplary embodiment of the present invention, the preservative is selected from sodium hydroxide.
As an alternative embodiment, the chelating agent is not particularly limited, and in the prior art, the chelating agent used for the gel in the medical field may be any.
As an exemplary embodiment of the present invention, the chelating agent is selected from EDTA.
In the present invention, the remaining component is water.
As a preferred embodiment of the present invention,
the hydrogel comprises the following components:
human being
As a more preferred embodiment of the present invention, the hydrogel comprises the following components:
in another aspect, the present invention further provides a method for preparing the above hydrogel, wherein the method comprises the following steps:
(1) preparation of solution A: dissolving heparin sodium, human serum albumin and mannitol in PBS solution, filtering, sterilizing, and storing at-20 deg.C;
(2) preparation of solution B: dissolving methyl hydroxybenzoate, propyl hydroxybenzoate and beta-cyclodextrin with ethanol and propylene glycol, and adding EDTA;
(3) preparation of solution C: dissolving carbomer with glycerol and propylene glycol, adding water, incubating at 60-100 deg.C for more than 1 hr, adding pH regulator to adjust pH to 7-9, autoclaving, and storing in dark place;
(4) mixing the solution A, B, C, adding hEGF, and mixing;
the solution A, B, C is prepared in no order, and can be used as it is or stored for reuse.
As in the above mentioned methods, there is no corresponding component in the formulation, and this step is omitted.
As a specific embodiment, the method for preparing the hydrogel of the present invention comprises the steps of:
1) preparation of solution A: weighing heparin sodium, human serum albumin and mannitol, dissolving in PBS solution, filtering with 0.22um filter, sterilizing, packaging, and freezing at-20 deg.C;
(2) preparation of solution B: weighing methyl hydroxybenzoate, propyl hydroxybenzoate and beta-cyclodextrin, respectively adding ethanol and propylene glycol, adding EDTA, and diluting with water to desired volume;
(3) preparation of solution C: weighing carbomer, adding glycerol and propylene glycol, stirring, adding water, incubating at 80 deg.C for more than 2 hr, adding 10mol NaOH, stirring, adjusting pH to 7-8, autoclaving, and storing in dark place;
(4) mixing the solution A, B, C, adding hEGF, and mixing.
In the present invention, human epidermal growth factor (human EGF, hEGF) is a polypeptide existing in various tissues of human body and having a wide range of biological activities, and is a single-chain polypeptide consisting of 53 amino acids, also known as human oligopeptide-1. Epidermal growth factor is widely used at present, such as ophthalmic cornea injury, corneal transplantation after cataract resection, skin burn and wound, traumatic skin ulcer, oral ulcer, gastrointestinal ulcer, glioma, squamous cell carcinoma and the like; it can also be made into pharmaceutical cosmetic containing EGF for promoting metabolism of epidermal cells.
However, the current method of locally spraying and applying growth factor solution does not exert the best effect in practical application, and faces various problems, such as low bioavailability, easy degradation and damage, limited permeability and the like (experimental research on promotion of deep II burn wound healing by gene gun-human basic fibroblast growth factor gene [ D ] of second university of military medical science, 2008).
In order to solve the problem that the epidermal growth factor is easily degraded by protein kinase in wounds, the invention firstly combines the components of the epidermal growth factor, heparin sodium, human serum albumin, carbomer, beta-cyclodextrin and the like.
The heparin sodium is a straight-chain anionic glycosaminoglycan, has biological activities of inhibiting fibrin deposition in tissues, inhibiting fibrocyte hyperplasia related to protease and the like, and is mainly used as an anticoagulant in clinic.
The human serum albumin is soluble protein with the most abundant content in human plasma, has a nutritional effect, belongs to functional protein absorbable by human skin, and can be used for nourishing wounds.
Wherein the carbomer is white loose powder and has strong hygroscopicity. The molecular structure of the gel contains 52-68% of acid groups, so the gel has certain acidity, and the pH of 1% aqueous dispersion is 2.5-3.5, and the gel can be neutralized by alkaline substances to form the gel. After the carbomer is neutralized to ionize the carboxyl, molecular chains are dispersed and stretched due to the mutual repulsion of negative charges, and the carbomer is in a great swelling state and has viscosity.
As an exemplary embodiment of the invention, the carbomer is carbomer 940.
The invention has the beneficial technical effects that:
1. in the technical scheme, the epidermal growth factor, the heparin sodium, the human serum albumin, the carbomer, the beta-cyclodextrin, the mannitol, the ethanol, the propylene glycol and other components are compatible for the first time to prepare the hydrogel, the wound healing effect of the hydrogel is obviously superior to that of a carbomer hydrogel control group and an untreated group, and the hydrogel has a good wound healing effect.
2. In the technical scheme, the hydrogel has stability, wherein the potency of the human epidermal growth factor is 85.2% of the original potency after the human epidermal growth factor is placed at 4 ℃ for 24 months; the titer is 81.5 percent of the original titer after being placed at 25 ℃ for 18 months.
Drawings
FIG. 1 is a wound model on day 0 (left: hydrogel control; middle: no treatment control; right: hydrogel + EGF);
FIG. 2 is wound healing on day 2 (left: hydrogel control; middle: no treatment control; right: hydrogel + EGF);
FIG. 3 is wound healing at day 4 (left: hydrogel control; middle: no treatment control; right: hydrogel + EGF);
FIG. 4 is wound healing at day 6 (left: hydrogel control; middle: no treatment control; right: hydrogel + EGF);
FIG. 5 wound healing at day 8 (left: hydrogel control; middle: no treatment control; right: hydrogel + EGF);
FIG. 6 wound healing at day 10 (left: hydrogel control; middle: no treatment control; right: hydrogel + EGF);
FIG. 7 is a graph showing the trend of the change of the wound area;
FIG. 8 shows the stability results of example 3.
Detailed Description
The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others of the concepts fall within the scope of the invention.
In the present invention, "%" represents a mass volume percentage in the system.
Example 1
TABLE 1 hydrogels
The configuration method of the combination B is as follows: the remaining combinations can be adjusted according to the concentration of each component.
Preparation of solution A:
weighing 0.4g of heparin sodium, 1g of human serum albumin and 1g of mannitol, dissolving in 40ml of PBS solution, filtering and sterilizing by using a 0.22um filter, subpackaging and freezing at-20 ℃ for later use.
Preparation of solution B:
weighing 1.67g of methyl paraben, 0.83g of propyl paraben and 1.5g of beta-cyclodextrin, respectively adding 10ml of ethanol and propylene glycol to 25ml, then adding a proper amount of EDTA and fixing the volume to 50ml with water.
Preparation of solution C:
weighing 1g of carbomer 940, adding 1ml of glycerol and 1ml of propylene glycol, stirring uniformly, adding 43ml of water, incubating at 80 ℃ for more than 2 hours, adding 230ul of 10mol NaOH per month, stirring uniformly, measuring the pH value to be about 7, autoclaving, and storing in a dark place.
Preparation of EGF hydrogel
Sucking 1ml of solution C, adding 0.8ml of solution A, 0.2ml of solution B, adding a proper amount of EGF solution (EGF is dissolved in PBS solution), finally fixing the volume to 4ml to make the final concentration of EGF be 200ng/ml, and uniformly mixing for use.
Example 2
Establishment of rat wound model
1. Purchasing female SD rats over 8 weeks of age for normal rearing on SPF scale;
2. injecting chloral hydrate into the abdominal cavity to anaesthetize the rat, and pulling out the back hair of the back of the rat;
3. marking three marks with the size of 1 multiplied by 1cm on the back by using a marker pen;
4. making trauma wound with scissors according to the mark, completely removing epidermis and dermis layers to expose submucosal tissue;
5. applying hydrogel control group and experimental group on the corresponding wound, and breeding in single cage;
6. rats were observed daily and wound healing was recorded and continued to be administered every 24 hours prior to wound healing and the rate of wound healing was compared for each group.
The results of the experiment are shown in FIGS. 1-7.
Wherein, the left side: hydrogel control group (same as experimental group except that it does not contain EGF); the middle part: no treatment control group (wound not treated); right side: experimental group (application example 1, combination B)
As can be seen from fig. 1-7, the experimental group (EGF + hydrogel) has a better promoting effect on the healing of the rat wound, and compared with the control group, the experimental group can rapidly promote the growth of the granulation tissue of the wound, and the wound healing speed is significantly faster than that of the control group. And the hydrogel has a good absorption effect, so that the wounds of the experimental group have no obvious tissue fluid exudation, and the existence of the components such as heparin, paraben ester and the like in the experimental group also reduces the risks of blood exudation and infection.
Example 3 stability test
The EGF gel of combination B of example 1 was stored at 25 ℃ and 4 ℃ respectively, sampled periodically and tested for EGF concentration in the gel by the elisa method.
The experimental results are shown in fig. 8, and the results show that the EGF gel is placed at 4 ℃ for 24 months, and the titer is 85.2% of the original titer; after being placed at 25 ℃ for 18 months, the potency of the gel is 81.5 percent of the original potency, which shows that the gel of the invention has good stability and can effectively exert curative effect.
Although the embodiments of the present invention have been disclosed and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, combinations and omissions may be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. A hydrogel for promoting wound healing, comprising: the hydrogel comprises the following components:
the pH of the hydrogel is 6-8.
2. The hydrogel of claim 1, wherein said hydrogel comprises the following components:
the hydrogel has a pH of 7-8.
3. The hydrogel of claim 1, wherein said hydrogel comprises the following components:
4. the hydrogel of claim 1, wherein the hydrogel further comprises any one of the following components:
0 to 5 percent of preservative
0 to 5 percent of pH regulator
Chelating agent 1-10 uM.
5. The hydrogel of claim 4, wherein the hydrogel comprises the following components:
0 to 0.5 percent of preservative
0 to 0.05 percent of pH regulator
Chelating agent 1-10 uM.
6. The hydrogel of claim 4, wherein the preservative is selected from one or more of parabens, benzoic acid, sodium benzoate, benzalkonium chloride, benzalkonium bromide, chlorobutanol, and sorbitol; preferably, the preservative is selected from methyl paraben and propyl paraben;
preferably, the pH regulator is selected from one or more of sodium hydroxide, hydrochloric acid, phosphate, triethanolamine, sodium bicarbonate, citric acid, citrate, boric acid and borate; more preferably, the pH adjusting agent is selected from sodium hydroxide;
preferably, the chelating agent is EDTA.
7. The hydrogel of claim 4, wherein the remaining component is water.
8. The hydrogel of claim 4, wherein the hydrogel comprises the following components:
preferably, the hydrogel comprises the following components:
9. the method for producing the hydrogel according to claim 8, comprising the steps of:
(1) preparation of solution A: dissolving heparin sodium, human serum albumin and mannitol in PBS solution, filtering, sterilizing, and storing at-20 deg.C;
(2) preparation of solution B: dissolving methyl hydroxybenzoate, propyl hydroxybenzoate and beta-cyclodextrin with ethanol and propylene glycol, and adding EDTA;
(3) preparation of solution C: dissolving carbomer with glycerol and propylene glycol, adding water, incubating at 60-100 deg.C for more than 1 hr, adding pH regulator to adjust pH to 6-8, autoclaving, and storing in dark place;
(4) mixing the solution A, B, C, adding hEGF, and mixing;
wherein the solution A, B, C is disposed in no order.
10. The method of claim 9, comprising the steps of:
(1) preparation of solution A: weighing heparin sodium, human serum albumin and mannitol, dissolving in PBS solution, filtering with 0.22um filter, sterilizing, packaging, and freezing at-20 deg.C;
(2) preparation of solution B: weighing methyl hydroxybenzoate, propyl hydroxybenzoate and beta-cyclodextrin, respectively adding ethanol and propylene glycol, adding EDTA, and diluting with water to desired volume;
(3) preparation of solution C: weighing carbomer, adding glycerol and propylene glycol, stirring, adding water, incubating at 80 deg.C for more than 2 hr, adding 10mol NaOH to adjust pH to 7-8, autoclaving, and storing in dark place;
(4) mixing the solution A, B, C, adding hEGF, and mixing.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115505566A (en) * | 2022-09-30 | 2022-12-23 | 国家卫生健康委科学技术研究所 | Acellular matrix material based on human-derived mesenchymal stem cells and preparation method thereof |
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