CN114246975B - Fish collagen wound repair sponge loaded with stem cells and preparation method thereof - Google Patents
Fish collagen wound repair sponge loaded with stem cells and preparation method thereof Download PDFInfo
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- A—HUMAN NECESSITIES
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- 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
- A61L15/42—Use of materials characterised by their function or physical properties
- A61L15/425—Porous materials, e.g. foams or sponges
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
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- A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
- A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
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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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- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
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- A61L15/42—Use of materials characterised by their function or physical properties
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
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Abstract
The invention discloses a stem cell-loaded fish collagen wound repair sponge and a preparation method thereof, wherein the preparation method comprises the following steps: preparing a fish collagen wound repair sponge, enabling the fish collagen wound repair sponge to absorb a cell complete culture medium, preparing a stem cell suspension, injecting stem cells into the fish collagen wound repair sponge and continuously providing the fish collagen wound repair sponge with the cell complete culture medium, co-culturing the stem cells and the fish collagen wound repair sponge, and performing mild dehydration treatment. According to the invention, the stem cells are loaded into the fish collagen wound repair sponge, so that the stem cells are distributed throughout the inside of the whole fish collagen wound repair sponge and grow, crawl and divide in the fish collagen wound repair sponge, and finally the inside of the whole fish collagen wound repair sponge is filled with the stem cells, so that the stem cells are induced and differentiated at the wound after the later-stage repair material is implanted, and the target function is achieved.
Description
Technical Field
The invention relates to a collagen wound repair sponge and a preparation method thereof, in particular to a stem cell-loaded fish collagen wound repair sponge and a preparation method thereof, and belongs to the technical field of wound repair materials.
Background
In recent years, with the progress of society and the acceleration of life pace, the number of patients with diabetes caused by overwork, irregular diet and the like is increasing year by year, the age of the patients tends to be low, the incidence rate of complications such as diabetic foot, chronic healing wound and the like caused by diabetes is also on the trend of increasing year by year, and the treatment of complications such as diabetic foot, chronic healing wound and the like is a problem to be solved clinically at present. In addition, repair of large-area skin injuries such as burns and wounds caused by various physical and chemical reasons is also a problem to be solved clinically at present.
At present, various dressing auxiliary treatments are mainly adopted for treating diabetic feet, chronic healing wounds and the like clinically, the treatment mode has long treatment period and relatively high treatment cost, meanwhile, patients have a lot of inconvenience, so that the economic burden and other burdens are brought to patients and families, how to accelerate the healing of the chronic healing wounds by a simple and easy-to-operate mode is always the direction of efforts of vast medical care workers and scientific research workers. Aiming at large-area skin injury, autologous skin transplantation is still the gold standard for clinical treatment, but the problems of survival rate of skin after autologous transplantation, secondary injury to organisms and the like greatly limit the application of the method, how to find a substitute material which can effectively survive and induce wound repair is the research and the effort direction of vast medical care workers and scientific research workers at present.
At present, the clinically used wound repair materials mainly comprise a plurality of sponges, dressings, gels, sprays, factors and the like, and the wound repair materials play a great role in various types of wound repair, but have common manifestations in auxiliary treatment of diabetic foot and chronic healing wounds and large-area skin injury repair. The most key point of skin injury repair is to recover blood supply, blood vessel ingrowth, cell differentiation and the like play important roles in the process, and if stable stem cells can be provided for a wound surface locally, the stem cells are differentiated towards the direction of cells promoting skin growth, so that the rapid healing of the wound surface is promoted. However, if the stem cells are only injected to the wound surface position, the stem cells quickly enter a circulatory system along with tissue fluid and cannot be positioned, so that the method for fixing the stem cells to the wound surface position in a targeted manner has important clinical value and clinical significance.
Disclosure of Invention
The invention aims to: provides a collagen wound repair sponge which can fix allogeneic stem cells or autologous stem cells at the wound position in a targeted manner so as to promote the wound to heal quickly and a preparation method thereof.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a preparation method of a fish collagen wound repair sponge loaded with stem cells is characterized by comprising the following steps:
step 1: preparing a fish collagen wound repair sponge;
and 2, step: enabling the fish collagen wound repair sponge to absorb a complete cell culture medium;
and 3, step 3: preparing a stem cell suspension;
and 4, step 4: injecting stem cells into the fish collagen wound repair sponge, and continuously providing a cell complete culture medium for the fish collagen wound repair sponge;
and 5: co-culturing stem cells and the fish collagen wound repair sponge;
step 6: and (4) mild dehydration treatment.
Preferably, in step 1, the method for preparing the fish collagen wound repair sponge specifically comprises the following steps:
(1) Taking a fish collagen water solution, and concentrating the concentration of the fish collagen to 0.4g/mL;
(2) Pouring the concentrated fish collagen aqueous solution into a freeze-drying tray of a freeze dryer, and putting the manufactured grid-shaped mold into the freeze-drying tray;
(3) Putting the freeze-drying tray into a freeze-drying machine, pre-freezing for 8h at-70 ℃, then freeze-drying for 48h at-70 ℃ according to the program, then gradually heating to room temperature, and taking out the freeze-drying tray;
(4) And taking the prepared fish collagen wound repair sponge out of the square, packaging and sterilizing.
Preferably, in step 2, when the weight of the fish collagen wound repair sponge reaches 15 to 21 times of the initial weight, the fish collagen wound repair sponge is transferred into a new culture bottle.
Preferably, in step 4, the method for injecting the stem cells into the fish collagen wound repair sponge specifically comprises the following steps: the prepared stem cell suspension is injected into a sterile syringe according to the proportion 1 x 10 5 Per cm 3 The density of the fish collagen is uniform and the fish collagen is injected into the wound repair sponge which has fully absorbed the complete cell culture medium.
Preferably, in step 4, the method for continuously providing the complete cell culture medium for the fish collagen wound repair sponge specifically comprises the following steps: slowly adding a cell complete culture medium into a culture bottle containing the fish collagen wound repair sponge, so that the liquid level of the cell complete culture medium reaches 1/4-1/3 of the height of the fish collagen wound repair sponge.
Preferably, in step 5, the co-cultivation method is as follows: placing the fish collagen wound repair sponge injected with the stem cells in a carbon dioxide incubator, co-culturing for 2 days at 37 ℃, then replacing a complete cell culture medium in a culture bottle, continuously culturing for 1-3 days at 37 ℃, and allowing the concentration of the stem cells in the fish collagen wound repair sponge to reach 1 x 10 6 Per cm 3 When the incubation is completed, the co-cultivation is completed.
Preferably, in step 6, the dehydration treatment method specifically comprises the following steps: and (3) performing mild dehydration treatment on the cultured repairing material at 37 ℃, and taking out the repairing material when the weight of the repairing material is reduced to 1/2-2/3 of the weight before dehydration.
A stem cell-loaded fish collagen wound repair sponge is characterized by being prepared by the method.
The invention has the advantages that:
1. the fish collagen wound repair sponge used in the invention has a large amount of hydroxyl and carboxyl on a peptide chain, so that the fish collagen wound repair sponge has good hydrophilicity and cell adhesion capability, and stem cells can grow, crawl and divide conveniently in the fish collagen wound repair sponge.
2. According to the invention, the stem cells are injected into the fish collagen wound repair sponge in an injection manner, so that the stem cells are spread inside the whole fish collagen wound repair sponge and grow, crawl and divide in the fish collagen wound repair sponge, and finally, the inside of the whole fish collagen wound repair sponge is filled with the stem cells, so that the stem cells are induced and differentiated at the wound after the later-stage repair material is implanted.
3. According to the invention, the cultured repair material is subjected to mild dehydration treatment, so that the pore diameter of the fish collagen wound repair sponge is effectively reduced, stem cells can be better fixed inside the fish collagen wound repair sponge, and the stem cells can conveniently perform a targeted function at the wound position after the repair material is implanted in the later period.
Drawings
FIG. 1 is an infrared absorption spectrum of a fish collagen wound repair sponge prepared by the invention;
FIG. 2 is a scanning electron micrograph of the repair material after dehydration;
FIG. 3 is a graph showing the results of staining of live and dead cells of a repair material before dehydration;
FIG. 4 is a fluorescent image of rat backs 7 days after implantation of a stem cell loaded fish collagen wound repair sponge at a back wound site;
FIG. 5 is a fluorescence image of rat backs 7 days after direct injection of stem cells at the dorsal wound site;
FIG. 6 is a graph of wound healing in rats 21 days after implantation of a stem cell loaded fish collagen wound repair sponge at the location of a chronic wound (1.5 cm diameter) induced by diabetes;
FIG. 7 is a graph of wound healing in rats 21 days after treatment of chronic diabetes-induced wounds (1.5 cm diameter) in rats without any treatment.
Detailed Description
The invention is described in detail below with reference to the figures and the embodiments.
First part, preparation of Fish collagen wound repair sponge
The preparation process of the fish collagen wound repair sponge used by the invention is as follows:
(1) Taking a fish collagen water solution, and concentrating the concentration of the fish collagen to 0.4g/mL by using a membrane filtration system;
(2) Pouring the concentrated fish collagen aqueous solution into a freeze-drying tray of a freeze dryer, wherein the liquid depth is 1cm (which can be adjusted within the range of 0.1-2 cm according to actual requirements), and then putting the prepared grid-shaped mold into the freeze-drying tray, wherein the side length of each grid is 1cm (which can be adjusted within the range of 0.5-30 cm according to actual requirements);
(3) Putting the freeze-drying tray into a freeze-drying machine, pre-freezing for 8h at-70 ℃, then freeze-drying for 48h at-70 ℃ according to the program, then gradually heating to room temperature, and taking out the freeze-drying tray;
(4) And taking the prepared fish collagen wound repair sponge out of the square, packaging and sterilizing.
Identifying the structure of the prepared fish collagen wound repair sponge, wherein the identification content and the identification process are as follows:
the natural type I collagen has specific infrared absorption band, and the absorption peak of the amide A band reflecting hydrogen bond stretching vibration absorption in N-H and O-H is 3400-3440 cm -1 Within a wavelength range. And (3) preparing the prepared fish collagen wound repair sponge into powder, carrying out infrared scanning on the powder by using a Fourier infrared spectrometer, and observing and judging the absorption band condition of the powder.
Through detection, the infrared absorption spectrum of the fish collagen wound repair sponge is shown in figure 1.
As can be found from the infrared absorption spectrogram shown in figure 1, the fish collagen wound repair sponge prepared by the invention is 3398cm -1 The position has a strong absorption peak, which indicates that the fish collagen wound repair sponge contains a large amount of N-H bonds and O-H bonds, and indirectly proves that a large amount of hydroxyl and carboxyl are arranged on a peptide chain of the fish collagen wound repair sponge.
The prepared fish collagen wound repair sponge has a large amount of hydroxyl and carboxyl on a peptide chain, so that the fish collagen wound repair sponge has good hydrophilicity and cell adhesion capability, and stem cells can grow, crawl and split at the wound more conveniently after being loaded on the fish collagen wound repair sponge.
The second part is that the wound repair sponge of fish collagen is loaded with stem cells
The process of loading stem cells on the fish collagen wound repair sponge specifically comprises the following steps:
1. complete culture medium for suction cells of fish collagen wound repair sponge
Placing the fish collagen wound repair sponge in a culture bottle A containing a cell complete culture medium under an aseptic environment, enabling the fish collagen wound repair sponge to fully absorb the cell complete culture medium, and transferring the fish collagen wound repair sponge into a culture bottle B for later use when the weight of the fish collagen wound repair sponge reaches 15-21 times of the initial weight.
2. Preparation of Stem cell suspensions
Culturing the allogeneic stem cells (or autologous stem cells) in a culture bottle C until the bottom of the culture bottle is more than 80% of the total volume, then digesting the allogeneic stem cells (or autologous stem cells) by using trypsin, centrifuging, taking cell precipitates, and finally preparing a stem cell suspension by using a cell complete culture medium for later use.
3. Injecting stem cells into the wound repair sponge of fish collagen
The prepared stem cell suspension is injected into a sterile syringe according to the proportion 1 x 10 5 Per cm 3 The cell complete culture medium is slowly added into a culture bottle B containing the fish collagen wound repair sponge, so that the liquid level of the cell complete culture medium reaches 1/4-1/3 of the height of the fish collagen wound repair sponge.
While continuously providing a complete cell culture medium for the fish collagen wound repair sponge, the stem cells are prevented from entering the culture medium in the bottle from the fish collagen wound repair sponge.
4. Co-culturing stem cells and fish collagen wound repair sponge
Placing the fish collagen wound repair sponge injected with the stem cells in a carbon dioxide incubator, co-culturing for 2 days at 37 ℃, then replacing a complete cell culture medium in a culture bottle B, continuously culturing for 1-3 days at 37 ℃, and allowing the concentration of the stem cells in the fish collagen wound repair sponge to reach 1 x 10 6 Per cm 3 Then, the fish collagen wound repair sponge (hereinafter referred to as repair material) loaded with stem cells is obtained, and the repair material is taken out for standby.
5. Dewatering
And (3) carrying out mild air drying dehydration treatment on the cultured repair material at 37 ℃, and taking out the repair material for later use when the weight of the repair material is reduced to 1/2-2/3 of the weight before dehydration.
And carrying out electron microscope scanning on the dehydrated repairing material. The scanning results are shown in fig. 2. The scan results of fig. 2 show that: a large number of stem cells are arranged on the surface and inside of the fish collagen wound repair sponge, and the stem cells are in creeping (pseudopodia extending), adhering and dividing states, and the growth state of the stem cells is good. The first part shows that the prepared fish collagen wound repair sponge can provide a good bracket for cell crawling and cell adhesion, and the cells can be well loaded on the fish collagen wound repair sponge.
When the repair material after dehydration is subjected to electron microscope scanning, the repair material before dehydration is also subjected to electron microscope scanning, and the scanning result shows that: compared with the fish collagen wound repair sponge before dehydration, the pore diameter of the fish collagen wound repair sponge is effectively reduced through mild dehydration treatment. After the pore size is reduced, the stem cells can be better fixed in the fish collagen wound repair sponge, and the stem cells can play a role in the target of the wound position after the later repair material is implanted.
In order to further verify the co-culture state of the stem cells and the fish collagen wound repair sponge, live and dead cell staining is carried out on the repair material before dehydration. The staining results are shown in FIG. 3.
The staining results in fig. 3 show: in the same visual field range, the surface and the inside of the repair material before dehydration are fully distributed with green fluorescence, and only a few red fluorescence exists. The fact that most of stem cells loaded on the fish collagen wound repair sponge are living cells and a few of stem cells are subjected to apoptosis is shown, and the fact that the fish collagen wound repair sponge prepared by the first part has good cell compatibility can induce the cells to perform crawling, adhesion, division and other behaviors on the surface and inside of the cells is also shown.
The third part is to verify the wound repair effect of the repair material
Experiment 1
Experimental groups: marking the cell nucleus of the stem cell by green fluorescence, then loading the stem cell on a fish collagen wound repair sponge according to the method recorded in the second part, then implanting the fish collagen wound repair sponge into the position of the back wound of a rat, and observing the fluorescence luminescence condition after 7 days.
Control group: the cell nucleus of the stem cell is marked by green fluorescence, then the stem cell is directly injected at the position of the wound surface on the back of the rat, and the fluorescence luminescence condition is observed after 7 days.
And (4) observing results: after 7 days, the fluorescence luminescence condition of the backs of the rats in the experimental group is shown in figure 4, and a large amount of green fluorescence is still left at the wound surface position of the rats; the fluorescence emission of the back of the control rat is shown in FIG. 5, and the wound site of the rat has no green fluorescence.
As can be seen from fig. 4 and 5, after the stem cells are loaded on the fish collagen wound repair sponge, the stem cells can be fixed to the wound position to play a role.
Experiment 2
Experimental groups: the fish collagen wound repair sponge loaded with stem cells was implanted in chronic wounds (diameter 1.5 cm) induced by diabetes in rats and the time required for the complete healing of the wounds in rats was recorded.
Control group: no treatment was performed on chronic wounds (diameter 1.5 cm) induced by diabetes in rats, and the healing of the wounds in rats in the control group was observed when the wounds in rats in the experimental group were all healed.
And (4) observing the results: on day 21, the wounds of all rats in the experimental group healed completely as shown in fig. 6, while, at this time, the wounds of around 1cm in diameter had not healed in all rats in the control group as shown in fig. 7.
Therefore, the repairing material provided by the invention can fix the stem cells at the position of the wound surface in a targeted manner, and provides stable stem cells for the wound surface locally, so that the rapid healing of the wound surface is promoted.
It should be noted that the above-mentioned embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the protection scope of the present invention.
Claims (4)
1. A preparation method of a fish collagen wound repair sponge loaded with stem cells is characterized by comprising the following steps:
step 1: preparing a fish collagen wound repair sponge, specifically, (1) taking a fish collagen water solution, and concentrating the concentration of fish collagen to 0.4g/mL; (2) Pouring the concentrated fish collagen aqueous solution into a freeze-drying tray of a freeze dryer, and putting the prepared grid-shaped mold into the freeze-drying tray; (3) Putting the freeze-drying tray into a freeze-drying machine, pre-freezing for 8h at-70 ℃, then freeze-drying for 48h at-70 ℃ according to the program, then gradually heating to room temperature, and taking out the freeze-drying tray; (4) Taking the prepared fish collagen wound repair sponge out of the square, packaging and sterilizing;
step 2: enabling the fish collagen wound repair sponge to absorb a complete cell culture medium;
and step 3: preparing a stem cell suspension;
and 4, step 4: injecting stem cells into the fish collagen wound repair sponge, continuously providing a cell complete culture medium for the fish collagen wound repair sponge, and specifically, injecting the prepared stem cell suspension into a sterile injector according to the proportion of 1 × 10 5 Per cm 3 The mixture is injected into the fish collagen wound repair sponge which has fully absorbed the cell complete culture medium, and the cell complete culture medium is slowly added into a culture bottle which contains the fish collagen wound repair sponge, so that the liquid level of the cell complete culture medium reaches 1/4-1/3 of the height of the fish collagen wound repair sponge;
and 5: co-culturing stem cells and the fish collagen wound repair sponge;
step 6: and (3) carrying out mild air drying dehydration treatment on the cultured repairing material at 37 ℃, and taking out the repairing material when the weight of the repairing material is reduced to 1/2-2/3 of the weight before dehydration.
2. The method for preparing a stem cell-loaded fish collagen wound repair sponge according to claim 1, wherein in step 2, the fish collagen wound repair sponge is transferred to a new culture flask when the weight of the fish collagen wound repair sponge reaches 15 to 21 times of the initial weight.
3. The method for preparing a stem cell-loaded fish collagen wound repair sponge according to claim 1, wherein in step 5, the co-culture method specifically comprises the following steps: fish gelatin injected with stem cellsPlacing the collagen wound repair sponge in a carbon dioxide incubator, co-culturing for 2 days at 37 ℃, then replacing a complete cell culture medium in a culture bottle, continuing culturing for 1-3 days at 37 ℃, and when the concentration of stem cells in the collagen wound repair sponge reaches 1 x 10 6 Per cm 3 When the incubation is completed, the co-cultivation is completed.
4. A stem cell loaded fish collagen wound repair sponge, prepared by the method of any one of claims 1 to 3.
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| CN104338175A (en) * | 2013-08-01 | 2015-02-11 | 上海华睿生物科技有限公司 | Preparing method of medical and sterile heat-resource-free fishskin collagen sponge |
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| CN108653193A (en) * | 2018-06-15 | 2018-10-16 | 大连医科大学附属第医院 | A kind of stem cell medicine sponge patch complex, preparation method and application for treating brain diseases |
| CN112745386A (en) * | 2021-02-08 | 2021-05-04 | 刘书乐 | Tilapia collagen sponge for traceless tissue damage repair and preparation method thereof |
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