CN116392579A - Cell fiber gel for promoting repair of diabetic skin injury and preparation method thereof - Google Patents
Cell fiber gel for promoting repair of diabetic skin injury and preparation method thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/14—Alkali metal chlorides; Alkaline earth metal chlorides
-
- 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
- A61K38/36—Blood coagulation or fibrinolysis factors
- A61K38/363—Fibrinogen
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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
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/482—Serine endopeptidases (3.4.21)
- A61K38/4833—Thrombin (3.4.21.5)
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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
- A61K47/30—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
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- 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
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21005—Thrombin (3.4.21.5)
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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Abstract
The invention relates to a cell fiber gel for promoting repair of diabetic skin injury and a preparation method thereof, which are used for solving the problem that no specific medicine exists in the existing treatment of diabetic skin injury. The cell fiber gel of the invention comprises the following raw materials: fibrinogen solution, wherein the concentration of fibrinogen is 23-30 mg/ml; thrombin cell suspension, said thrombin cell suspensionThe cell suspension comprises dental pulp stem cells, thrombin and calcium chloride, and the concentration of the dental pulp stem cells is 5 multiplied by 10 4 ～1×10 7 The concentration of thrombin is less than 45IU/mL, and the concentration of calcium chloride is 0-5 mmol/L; wherein the volume ratio of the fibrinogen solution to the thrombin cell suspension is 1:1.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a cell fiber gel for promoting repair of diabetic skin injury and a preparation method thereof.
Background
The skin injury caused by diabetes is a common wound surface which is difficult to heal due to diabetes, and the wound surface is not healed after the skin defect of a patient due to insufficient blood supply, so that infection is gradually aggravated, and finally tissue necrosis or amputation is caused. According to the global epidemiological system review and meta analysis data of the university of Nanjing, the prevalence of diabetic foot ulcers is 6.3% worldwide (about 2900 tens of thousands), and the prevalence of China is 4.1% (410 tens of thousands). It is counted that every 20 seconds worldwide there is amputation of one patient with diabetic foot, and the treatment cost of diabetic foot is about one third of the treatment cost of the whole diabetes, which causes a great public health problem due to the heavy economic burden brought to many families and society.
At present, no specific medicine is used for treating the skin injury of diabetes, and no medicine capable of remarkably promoting the healing of the wound of the diabetes is used for removing systemic metabolism improving medicines (sugar control) and antibiotic medicines for inhibiting infection. The development of safe and effective medicaments for repairing diabetic skin lesions remains an unmet clinical need.
Disclosure of Invention
In view of the above analysis, the embodiment of the invention aims to provide a cell fiber gel for promoting the repair of diabetic skin injury and a preparation method thereof, which are used for solving the problem that no specific medicine exists in the existing treatment of diabetic skin injury.
In one aspect, the invention provides a cell fiber gel for promoting repair of diabetic skin injury, which comprises the following raw materials:
fibrinogen solution, wherein the concentration of fibrinogen is 23-30 mg/ml;
a thrombin cell suspension comprising dental pulp stem cells, thrombin and calcium chloride, wherein the dental pulp stem cells have a concentration of 5 x 10 4 ～1×10 7 The concentration of thrombin is less than 45IU/mL, and the concentration of calcium chloride is 0-5 mmol/L;
wherein the volume ratio of the fibrinogen solution to the thrombin cell suspension is 1:1.
Further, the dental pulp stem cells are human dental pulp stem cells.
Further, the preparation method of the fibrinogen comprises the following steps:
s101: adding the S/D mother solution into the Cohn I solution, and stirring to obtain an inactivated Cohn I solution;
s102: precipitating the inactivated Cohn I solution obtained in the step S101 by using low-temperature ethanol, and centrifuging to obtain fibrinogen precipitate;
s103: and (3) washing the fibrinogen precipitate obtained in the step S102 by using a washing liquid, and packaging.
Further, the step S101 includes the steps of:
s101-1: dissolving frozen Cohn I to obtain Cohn I solution;
s101-2: carrying out virus inactivation by adopting S/D, adding the S/D mother liquor into the Cohn I solution, wherein the volume ratio of the S/D mother liquor to the Cohn I solution is 1:9, and stirring for 6 hours at the temperature of 24+/-1 ℃ to obtain an inactivated Cohn I solution;
in the step S102, firstly cooling the inactivated Cohn I solution obtained in the step S101-2 to below 0 ℃, then dropwise adding a 50% ethanol solution precooled at the temperature of minus 20 ℃ while stirring until the final concentration of the dropwise adding ethanol is 8%, continuing stirring for 30min after the dropwise adding is finished, standing for 1h, centrifuging at the temperature of minus 2 ℃ for 15min, discarding the supernatant, and collecting fibrinogen precipitate;
the step S103 includes the steps of:
s103-1: the fibrinogen precipitate obtained in the step S102 and the washing liquid are stirred and dissolved at the temperature of 27 ℃ according to the mass ratio of 1:9, and then the step S102 is repeated for one time, and the washing is carried out twice;
s103-2: and (3) stirring and dissolving the fibrinogen precipitate obtained in the step S103-1 and the fibrinogen solution according to the mass ratio of 1:4 at the temperature of 27 ℃ for packaging.
Further, the preparation method of thrombin comprises the following steps:
s201: obtaining a plasma-adsorbed gel;
s202: washing and eluting the gel for adsorbing the plasma obtained in the step S201, and filtering and concentrating the eluent;
s203: inactivating viruses by adopting an S/D method;
s204: prothrombin activation;
s205: gel adsorption and purification;
s206: ultrafiltering, concentrating, and packaging;
s207: and inactivating viruses by a dry heat method.
Further, in the step S201, after the gel is swelled, balancing is performed by using a balancing solution, and the balancing solution is added into the cryoprecipitated plasma, and stirred and adsorbed for 30-60 min at the temperature of 2-15 ℃ to obtain the gel for adsorbing the plasma;
in the step S202, gel adsorbing plasma is washed by using balance liquid and washing liquid in sequence, then eluting is performed by using eluent, and the eluent is collected, filtered and concentrated by ultrafiltration until the protein content is 20+/-10 g/L;
in the step S203, the pH of the product is regulated to 7.00+/-0.20, in the stirring process, S/D solution is added to ensure that the final concentration of the detergent in the product is 1 percent and the final concentration of the organic solvent is 0.3 percent, then the pH of the product is regulated to 7.00+/-0.20 again, and the mixture is stirred and inactivated for 6 hours at the temperature of 24.0+/-2 ℃;
in the step S204, after virus inactivation is finished, the product conductance is regulated to 6-16 ms/cm, 1+/-0.1 mol/L calcium chloride solution is added to enable the final concentration to reach 7mmol/L, the temperature is raised to 37 ℃, the product temperature is kept at 36-38 ℃ for stirring reaction for 1-3 h, then the temperature is reduced to 24 ℃, and the product temperature is kept at 24.0-26.0 ℃ for stirring reaction for 24-30 h;
the step S205 includes the following steps:
s205-1: the gel is firstly washed by 2.0mol/L sodium chloride solution, 1.0 mol/L sodium hydroxide solution and 0.1mol/L sodium chloride solution in sequence, and then is balanced by a balancing solution, and then can be put on a column;
s205-2: adjusting the pH of the product to 6.4-6.6, filtering by a microporous filter membrane with the thickness of 0.22 mu m, loading to a column for adsorption, and controlling the loading amount to be less than 10 times of the volume of the gel column bed;
s205-3: after the sample loading is finished, washing gel by using balance liquid, washing the gel by using washing liquid, eluting the gel by using eluent, collecting the eluent according to a chromatographic chart, and adding 60ml of 1+/-0.1 mol/L calcium chloride solution, 2g of human serum albumin, 5g of sucrose and 3g of glycine into each liter of product after measuring the volume;
the step S206 includes the steps of:
s206-1: dialyzing the product prepared in the step S205-3 in a cold storage at 4 ℃ overnight, sampling to determine the titer of thrombin, diluting the product with dialysate to make the titer of thrombin less than 550IU/ml, supplementing human serum albumin to 5g/L, and controlling the pH of the product to be 6.3-7.6;
s206-2: packaging the above products, freeze-drying at-60deg.C under vacuum degree of 0.001mbar for about 48 hr;
in the step S207, the freeze-dried human thrombin sample obtained in the step S206-2 is placed in a water bath box, a thermometer is placed at multiple points, timing is started when the temperature reaches 99 ℃, the temperature is recorded every 5min, the water bath temperature is controlled within the range of 99-100 ℃ in the inactivation process, and heating is kept for 30min.
In another aspect, the invention provides a method for preparing a cell fiber gel for promoting repair of diabetic skin injury, which is used for preparing the cell fiber gel and comprises the following steps:
step 1: respectively preparing fibrinogen and thrombin into a fibrinogen solution and a thrombin solution, wherein the concentration of the fibrinogen solution is 23-30 mg/mL, and the concentration of the thrombin solution is less than 45U/mL;
step 2: sequentially adding calcium chloride and dental pulp stem cell solution into the thrombin solution to obtain thrombin cell suspension, wherein the concentration of calcium chloride in the thrombin cell suspension is 0-5 mmol/L, and the concentration of dental pulp stem cells is 5 multiplied by 10 4 ～1×10 7 individual/mL;
step 3: mixing the fibrinogen solution with the thrombin cell suspension according to the volume ratio of 1:1, incubating for 1h at 37 ℃, and culturing for 12h in a culture medium containing 10% fetal bovine serum to obtain the cell fiber gel for promoting the repair of the diabetic skin injury.
Further, in the step 1, the fibrinogen is pretreated before being prepared into a solution, and the pretreatment process is as follows: the fibrinogen is pre-frozen overnight at the temperature of-70 ℃, then placed in a freeze dryer for vacuum freeze drying, the working condition is that a cold trap is stable at-60 ℃, the vacuum degree is 0.001mbar, and the freeze drying time is 72 hours.
Further, in the step 1, the thrombin is pretreated before being configured into a solution, and the pretreatment process is as follows: the thrombin solution was lyophilized at 60℃under a vacuum of 0.001mbar for 48h.
Further, the solvent of all the solutions was distilled water having a pH of 7 and subjected to autoclaving.
Further, in the step 3, the thickness of the single fibrin hydrogel is less than or equal to 2mm.
Compared with the prior art, the invention has at least one of the following beneficial effects:
(1) HE staining results show that the diabetic skin injury part treated by the cell fiber gel of the invention presents a complete epidermis-dermis-subcutaneous tissue structure and a new hair follicle appears;
(2) The Masson dyeing result shows that the diabetic skin injury part treated by the cell fiber glue has more collagen tissues and is similar to normal skin;
(3) The cell fiber gel can obviously promote the dental pulp stem cells to secrete the growth factor LIF, SCF, HGF, VEGF-A, can obviously promote the repair of the diabetic skin injury, can increase the wound healing speed, and can achieve the healing efficiency of 5.8% on the 2 nd day; on day 7, the healing efficiency can reach 47.2%; on day 12, the healing efficiency can reach 74.9%.
In the invention, the technical schemes can be mutually combined to realize more preferable combination schemes. Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention may be realized and attained by the structure particularly pointed out in the written description and drawings.
Drawings
The drawings are only for purposes of illustrating particular embodiments and are not to be construed as limiting the invention, like reference numerals being used to refer to like parts throughout the several views.
FIG. 1 is a graph of the ability of example and control groups to heal diabetic wounds (wherein 1 represents example 1,2 represents example 2,3 represents example 3,4 represents example 4,5 represents control) in the detailed description;
FIG. 2 is a photograph of skin wound surface of each diabetic mouse on days 0, 2, 7, 12 after operation of the blank group and the control group in the specific embodiment;
FIG. 3 is a bar graph of wound healing rate for each diabetic mouse on days 0, 2, 7, 12 post-surgery for the blank and control groups in accordance with one embodiment;
FIG. 4 is a photograph of skin wound surface of each diabetic mouse on days 0, 2, 7, 12 after operation of the blank group and the example group in the specific embodiment;
FIG. 5 is a bar graph of wound healing rate for each diabetic mouse on days 0, 2, 7, 12 post-surgery for the blank and example groups in the detailed description;
FIG. 6 is a graph of HE staining of whole-layer skin tissue paraffin sections of diabetic mice in a control group, an example group and a normal group according to the embodiment;
FIG. 7 shows fluorescence staining of CK14 paraffin sections of whole-layer skin tissue of diabetic mice in a control group, an example group and a normal group in the specific embodiment;
fig. 8 is a Masson stain of whole-layer skin tissue paraffin sections of diabetic mice in the control group, the example group, and the normal group in the specific embodiment.
Detailed Description
The following detailed description of preferred embodiments of the invention is made in connection with the accompanying drawings, which form a part hereof, and together with the description of the embodiments of the invention, are used to explain the principles of the invention and are not intended to limit the scope of the invention.
In describing embodiments of the present invention, it should be noted that, unless explicitly stated and limited otherwise, the term "coupled" should be interpreted broadly, for example, as being fixedly coupled, as being detachably coupled, as being integrally coupled, as being mechanically coupled, as being electrically coupled, as being directly coupled, as being indirectly coupled via an intermediate medium. The specific meaning of the above terms in the present invention can be understood by those of ordinary skill in the art according to the specific circumstances.
The terms "top," "bottom," "above … …," "below," and "on … …" are used throughout the description to refer to the relative positions of components of the device, such as the relative positions of the top and bottom substrates inside the device. It will be appreciated that the devices are versatile, irrespective of their orientation in space.
The working surface of the invention can be a plane or a curved surface, and can be inclined or horizontal. For convenience of explanation, the embodiments of the present invention are placed on a horizontal plane and used on the horizontal plane, and thus "up and down" and "up and down" are defined.
The invention discloses a cell fiber gel (hereinafter referred to as cell fiber gel) for promoting repair of diabetic skin injury, which comprises the following raw materials:
a fibrinogen solution having a fibrinogen concentration of 23 to 30mg/ml, preferably 27mg/ml;
a thrombin cell suspension comprising dental pulp stem cells, thrombin and calcium chloride, wherein the dental pulp stem cells have a concentration of 5 x 10 4 ～1×10 7 Each mL (preferably, the concentration of the dental pulp stem cells is 5.55X10) 6 A concentration of thrombin of less than 45IU/mL (preferably, a concentration of thrombin of 5 IU/mL), and a concentration of calcium chloride of 0 to 5mmol/L;
wherein the volume ratio of the fibrinogen solution to the thrombin cell suspension is 1:1.
It should be noted that, the ranges referred to in the present invention include the end values at both ends of the range.
Preferably, the solvent of the fibrinogen solution and thrombin cell suspension is distilled water, and the distilled water is adjusted to pH 7, and subjected to autoclaving, preferably distilled water is boiled at 121.3 ℃ for 20min.
Preferably, the dental pulp stem cells are human dental pulp stem cells. In this example, human dental pulp stem cells were derived from Beijing Sanyou and Zebiotech Inc. (4-6 passages) gifts.
The storage modulus of the cell fiber gel is in the range of 800-1200 Pa, and the cell fiber gel obtained beyond the range of the invention has no beneficial effect as the cell fiber gel.
According to a preferred embodiment of the present invention, the method for preparing fibrinogen comprises the steps of:
s101: adding the S/D mother solution into the Cohn I solution, and stirring to obtain an inactivated Cohn I solution;
s102: precipitating the inactivated Cohn I solution obtained in the step S101 by using low-temperature ethanol, and centrifuging to obtain fibrinogen precipitate;
s103: and (3) washing the fibrinogen precipitate obtained in the step S102 by using a washing liquid, and packaging.
Specifically, the step S101 includes the steps of:
s101-1: dissolving frozen Cohn I (Cohn's component I) to obtain a Cohn I solution;
s101-2: performing virus inactivation by an organic solvent/detergent method (S/D);
slowly adding the S/D mother solution into the Cohn I solution, wherein the volume ratio of the S/D mother solution to the Cohn I solution is 1:9, and stirring for 6 hours at the temperature of 24+/-1 ℃ to obtain the inactivated Cohn I solution. This step is capable of inactivating the enveloped viruses that may be present in the Cohn I solution. Illustratively, the organic solvent in the S/D mother liquor is tributyl phosphate (TNBP) and the detergent is polysorbate-80 (Tween-80).
In the step S102, the inactivated Cohn I solution obtained in the step S101 is slowly cooled to below 0 ℃, 50% ethanol solution precooled at-20 ℃ is slowly dripped, stirring is carried out while dripping until the final concentration of ethanol is 8%, stirring is continued for 30min after dripping is finished, standing is carried out for 1h, centrifuging is carried out at-2 ℃ for 15min after 1h, 8000g/min centrifuging is carried out, supernatant is discarded, and fibrinogen precipitate is collected.
The step S103 includes the steps of:
s103-1: the fibrinogen precipitate obtained in the step S102 and the washing liquid are stirred and dissolved at the temperature of 27 ℃ according to the mass ratio of 1:9, and then the step S102 is repeated for one time, and the washing is carried out for two times;
s103-2: the fibrinogen precipitate obtained in the step S103-1 and the fibrinogen solution were dissolved by stirring at 27℃in a mass ratio of 1:4, and were packaged in 25 ml/bottle.
Illustratively, in the step S103, the washing solution includes sodium citrate, sodium chloride, lysine hydrochloride, sucrose, tromethamine and water, wherein the mass fraction of sodium citrate is 0.8%, the concentration of sodium chloride is 0.1mol/L, the mass fraction of lysine hydrochloride is 0.44%, the mass fraction of sucrose is 1.25%, and the mass fraction of tromethamine is 0.27%. The fibrinogen dissolving solution comprises sodium citrate, sodium chloride, lysine hydrochloride, glycine, tromethamine and water, wherein the mass fraction of the sodium citrate is 0.8%, the concentration of the sodium chloride is 0.1mol/L, the mass fraction of the lysine hydrochloride is 0.44%, the mass fraction of the glycine is 0.1%, and the mass fraction of the tromethamine is 0.27%.
Preferably, the packaged fibrinogen solution is pre-frozen overnight at-70 ℃, then placed in a freeze dryer for vacuum freeze drying under the working conditions of cold trap stabilization at-60 ℃ and vacuum degree of 0.001mbar, and freeze drying time of 72h for use. Therapeutic assays were performed on fibrinogen prepared by the method described above: the fibrinogen concentration activity is measured, the fibrinogen clotting activity is 18-24 s, which is far smaller than the fibrinogen clotting activity within 60s specified in the pharmacopoeia of the people's republic of China (three, 2020 edition); the purity of the fibrinogen is measured to be 80% -85.62%, which is far higher than the purity of the fibrinogen of more than 70% specified in pharmacopoeia of the people's republic of China (three, 2020 edition). The fibrinogen prepared by the method has high purity and good coagulation activity, and the quality of the fibrinogen is far higher than the standard specified in the pharmacopoeia of the people's republic of China.
According to a preferred embodiment of the present invention, the method for preparing thrombin comprises the steps of:
s201: obtaining a plasma-adsorbed gel; preferably, a DEAE dextran gel is obtained that adsorbs plasma;
s202: washing and eluting the gel for adsorbing the plasma obtained in the step S201, and filtering and concentrating the eluent; preferably, the DEAE sephadex obtained in S201 and adsorbed to the plasma is washed and eluted, and the eluent is filtered and concentrated;
s203: inactivating viruses by adopting an S/D method;
s204: prothrombin activation;
s205: gel adsorption and purification; preferably, sulfopropyl agarose gel adsorption purification;
s206: ultrafiltering, concentrating, and packaging;
s207: and inactivating viruses by a dry heat method.
Specifically, in the step S201, after the gel is swelled, the gel is equilibrated with an equilibration solution, and added into cryoprecipitated plasma (preferably, cryoprecipitated plasma is human cryoprecipitated plasma), and the cryoprecipitated plasma is stirred and adsorbed for 30 to 60 minutes at a temperature of 2 to 15 ℃ to obtain the gel for adsorbing plasma. Illustratively, the gel is a DEAE Sephadex A-50 (diethylaminoethyl-dextran A-50) gel.
Illustratively, in the step S201, the balancing solution includes sodium citrate, sodium chloride and water, wherein the concentration of the sodium citrate is 0.01mol/L, the concentration of the sodium chloride is 0.1mol/L, and the pH is 7.60±0.20.
Preferably, the gel in the step S201 is DEAE dextran.
In the step S202, the gel that adsorbs plasma is washed with the equilibration solution and the washing solution in sequence (preferably, the DEAE sephadex that adsorbs plasma is washed with the equilibration solution and the washing solution in sequence), and then eluted with the eluent, and the eluent is collected, filtered, and concentrated by ultrafiltration until the protein content is 20±10g/L. Preferably, the gel absorbing the plasma is washed for 1 to 3 times by using the balance liquid, and the volume ratio of the gel absorbing the plasma to the balance liquid is 1 to 2:1; then washing the gel adsorbed with plasma for 4-6 times by using a washing liquid, wherein the volume ratio of the gel to the plasma is 1-2:1; finally, eluting the gel for adsorbing the plasma for 1 to 3 times by using eluent, wherein the volume ratio of the eluent to the gel is 1 to 2:1. The eluate was collected and filtered with a 0.22 μm filter membrane, and the filtered eluate was concentrated to a protein content of 20.+ -. 10g/L by ultrafiltration using a membrane package having a molecular weight of 10 k.
Illustratively, in the step S202, the balancing solution includes sodium citrate, sodium chloride and water, wherein the concentration of sodium citrate is 0.01mol/L, the concentration of sodium chloride is 0.1mol/L, and the pH is 7.60±0.20; the washing liquid comprises sodium citrate, sodium chloride and water, wherein the concentration of the sodium citrate is 0.01mol/L, the concentration of the sodium chloride is 0.2mol/L, and the pH is 7.00+/-0.20; the eluent comprises sodium citrate, sodium chloride and water, wherein the concentration of the sodium citrate is 0.01mol/L, the concentration of the sodium chloride is 0.5mol/L, and the pH is 7.00+/-0.20.
In the step S203, the pH of the product (i.e., the concentrated solution obtained in the step S202) is adjusted to 7.00 + -0.20, and during the stirring, the S/D solution is slowly added to make the final concentration of the detergent in the product 1% and the final concentration of the organic solvent 0.3%, and then the pH of the product is re-calibrated to 7.00 + -0.20, and the mixture is stirred and inactivated at 24.0 + -2 ℃ for 6 hours. The step has stable inactivating capability on the lipid-enveloped virus of the thrombin preparation intermediate solution, and is not easy to denature protein. Illustratively, in the S/D (organic solvent/detergent) solution, the organic solvent is tributyl phosphate (TNBP) and the detergent is Tween-80 (Tween-80).
In the step S204, after virus inactivation is finished, the electric conduction of the product (namely the sterilized solution obtained in the step S203) is regulated to 6-16 ms/cm (25 ℃), 1+/-0.1 mol/L calcium chloride solution is added to enable the final concentration to reach 7mmol/L, the temperature is raised to 37 ℃, the product temperature is kept at 36-38 ℃ for stirring reaction for 1-3 h, then the temperature is reduced to 24 ℃, and the product temperature is kept at 24.0-26.0 ℃ for stirring reaction for 24-30 h.
The step S205 includes the following steps:
s205-1: the gel is firstly washed by 2.0mol/L sodium chloride solution, 1.0 mol/L sodium hydroxide solution and 0.1mol/L sodium chloride solution in sequence, and then is balanced by a balancing solution, and then can be put on a column; that is, before the product (the activated liquid obtained in the step S204) is put on the column, the gel is washed and equilibrated before being put on the column; preferably, the gel is an SP Sepharose F.F gel;
s205-2: adjusting the pH of the product (the activated liquid obtained in the step S204) to 6.4-6.6, filtering by a microporous filter membrane with the thickness of 0.22 mu m, and then loading the product to a column for adsorption, wherein the loading amount is controlled below 10 times of the volume of a gel column bed;
s205-3: after the sample loading is finished, washing gel 4-6 times of column bed volume by using balance liquid, washing gel 2-4 times of column volume by using washing liquid, eluting gel 1-3 times of column bed volume by using eluent, collecting eluent according to a chromatographic chart, and adding 60ml of calcium chloride solution with the concentration of 1+/-0.1 mol/L, 2g of human serum albumin, 5g of sucrose and 3g of glycine into each liter of product after measuring the volume.
Illustratively, in the step S205, the balancing solution includes sodium gluconate, sodium chloride and water, wherein the concentration of sodium gluconate is 40mmol/L, the concentration of sodium chloride is 10mmol/L, and the pH is 6.5±0.2; the washing liquid comprises sodium gluconate, sodium chloride and water, wherein the concentration of the sodium gluconate is 150mmol/L, the concentration of the sodium chloride is 70mmol/L, and the pH is 6.5+/-0.2; the eluent comprises sodium gluconate, sodium chloride and water, wherein the concentration of the sodium gluconate is 150mmol/L, the concentration of the sodium chloride is 200mmol/L, and the pH is 6.5+/-0.2.
Preferably, the gel in the step S205 is sulfopropyl agarose gel.
The step S206 includes the steps of:
s206-1: dialyzing the product prepared in the step S205 in a cold storage at 4 ℃ overnight, sampling to determine the titer of thrombin, diluting the product with dialysate to ensure that the titer of thrombin is 550IU/ml, supplementing human serum albumin to 5g/L, and controlling the pH of the product to be 6.3-7.6;
s206-2: the above product was sub-packaged at 2 ml/min and lyophilized at-60deg.C under vacuum of 0.001mbar for about 48h for use.
Illustratively, the dialysate of step 206 includes calcium chloride, sodium gluconate, sucrose, glycine, sodium chloride, and water, wherein the concentration of calcium chloride is 60mmol/L, the concentration of sodium gluconate is 150mmol/L, the concentration of sucrose is 5g/L, the concentration of glycine is 3g/L, the concentration of sodium chloride is 80mmol/L, and the pH is 6.3-6.8.
In the step S207, the freeze-dried human thrombin sample obtained in the step S206 is placed in a water bath box, a thermometer is placed at multiple points, timing is started when the temperature reaches 99 ℃, the temperature is recorded every 5 minutes, the water bath temperature is controlled within the range of 99-100 ℃ in the inactivation process, and heating is kept for 30 minutes. This step is to perform virus inactivation, and the blood product must have 2 steps of virus inactivation/removal process during the production process, except for albumin. The method for inactivating the virus has inactivation effect on lipid-coated virus and non-lipid-coated virus, and has the advantages of no impurity introduced in the inactivation process, pollution avoidance and high safety.
According to thrombin samples and related verification results, the prepared human thrombin accords with the quality requirements specified in the pharmacopoeia of the people's republic of China (2020 edition, three parts).
The invention also discloses a preparation method of the cell fiber gel for promoting the repair of the diabetic skin injury, which can be used for preparing the cell fiber gel of the invention, and comprises the following steps:
step 1: fibrinogen and thrombin are respectively prepared into fibrinogen solution and thrombin solution, the concentration of the fibrinogen solution is 23-30 mg/mL, and the concentration of the thrombin solution is less than 45U/mL.
The cell fiber gel formed by taking the fibrinogen solution and the thrombin solution as raw materials has a certain jelly-like shape, and the hydrogel which is not atrophic after being picked out has a hydrogel colloid with stronger water retention capacity and uniform and stable shape.
Step 2: sequentially adding calcium chloride and dental pulp stem cell solution into the thrombin solution to obtain thrombin cell suspension, wherein the concentration of calcium chloride in the thrombin cell suspension is 0-5 mmol/L, and the concentration of dental pulp stem cells is 5 multiplied by 10 4 ～1×10 7 And each mL.
The addition of calcium ions can obviously thicken the fiber of the fiber gel, increase the pore diameter and promote the formation of the cell fiber gel.
Step 3: mixing the fibrinogen solution with the thrombin cell suspension according to the volume ratio of 1:1, incubating for 1h at 37 ℃, and culturing for 12h in a culture medium containing 10% fetal bovine serum to obtain the cell fiber gel for promoting the repair of the diabetic skin injury.
Preferably, in the step 3, the thickness of the single fibrin hydrogel is less than or equal to 2mm.
The solvents of all the above solutions were distilled water, and the distilled water was adjusted to pH 7, and autoclaved.
In the step 1, fibrinogen and thrombin are pretreated. Preferably, the fibrinogen and thrombin are subjected to freeze-drying treatment, in this embodiment, the fibrinogen is pretreated before being prepared into a solution, and the pretreatment process is as follows: the fibrinogen is pre-frozen overnight at the temperature of-70 ℃, then placed in a freeze dryer for vacuum freeze drying, the working condition is that a cold trap is stable at-60 ℃, the vacuum degree is 0.001mbar, and the freeze drying time is 72 hours. The thrombin is pretreated before being prepared into a solution, and the pretreatment process comprises the following steps: the thrombin solution was lyophilized at 60℃under a vacuum of 0.001mbar for 48h.
The preparation method of fibrinogen and thrombin in the step 1 is preferably the method described above.
Example 1 a method for preparing a cell sap for promoting repair of diabetic skin lesions, comprising the steps of:
step 1: preparing fibrinogen and thrombin into a fibrinogen solution and a thrombin solution respectively, wherein the concentration of the fibrinogen solution is 27mg/mL, and the concentration of the thrombin solution is 5I U/mL;
step 2: sequentially adding calcium chloride and dental pulp stem cell solution into the thrombin solution to obtain thrombin cell suspension, wherein the concentration of calcium chloride in the thrombin cell suspension is 0mmol/L (i.e. no calcium chloride is added), and the concentration of dental pulp stem cells is 2.8X10 × 6 individual/mL;
step 3: mixing the fibrinogen solution with the thrombin cell suspension according to the volume ratio of 1:1, incubating for 1h at 37 ℃, and culturing for 12h in a culture medium containing 10% fetal bovine serum to obtain the cell-fiber gel for promoting the repair of the diabetic skin injury.
Example 2 a method of preparing a cell sap for promoting repair of diabetic skin lesions comprising the steps of:
step 1: preparing fibrinogen and thrombin into fibrinogen solution and thrombin solution respectively, wherein the concentration of the fibrinogen solution is 23mg/mL, and the concentration of the thrombin solution is 5I U/mL;
step 2: sequentially adding calcium chloride and dental pulp stem cell solution into the thrombin solution to obtain thrombin cell suspension, wherein the concentration of calcium chloride in the thrombin cell suspension is 5mmol/L, and the concentration of dental pulp stem cells is 1.4X10 5 individual/mL;
step 3: mixing the fibrinogen solution with the thrombin cell suspension according to the volume ratio of 1:1, incubating for 1h at 37 ℃, and culturing for 12h in a culture medium containing 10% fetal bovine serum to obtain the cell-fiber gel for promoting the repair of the diabetic skin injury.
Example 3 a method of preparing a cell sap for promoting repair of diabetic skin lesions comprising the steps of:
step 1: preparing fibrinogen and thrombin into fibrinogen solution and thrombin solution respectively, wherein the concentration of the fibrinogen solution is 30mg/mL, and the concentration of the thrombin solution is 5I U/mL;
step 2: sequentially adding calcium chloride and dental pulp stem cell solution into the thrombin solution to obtain thrombin cell suspension, wherein the concentration of calcium chloride in the thrombin cell suspension is 0mmol/L (i.e. no calcium chloride is added), and the concentration of dental pulp stem cells is 1.4X10% 5 individual/mL;
step 3: mixing the fibrinogen solution with the thrombin cell suspension according to the volume ratio of 1:1, incubating for 1h at 37 ℃, and culturing for 12h in a culture medium containing 10% fetal bovine serum to obtain the cell-fiber gel for promoting the repair of the diabetic skin injury.
Example 4 a method of preparing a cell sap for promoting repair of diabetic skin lesions comprising the steps of:
step 1: preparing fibrinogen and thrombin into a fibrinogen solution and a thrombin solution respectively, wherein the concentration of the fibrinogen solution is 27mg/mL, and the concentration of the thrombin solution is 15I U/mL;
step 2: sequentially adding calcium chloride and dental pulp stem cell solution into the thrombin solution to obtain thrombin cell suspension, wherein the concentration of calcium chloride in the thrombin cell suspension is 0mmol/L (i.e. no calcium chloride is added), and the concentration of dental pulp stem cells is 1.4X10% 5 individual/mL;
step 3: mixing the fibrinogen solution with the thrombin cell suspension according to the volume ratio of 1:1, incubating for 1h at 37 ℃, and culturing for 12h in a culture medium containing 10% fetal bovine serum to obtain the cell-fiber gel for promoting the repair of the diabetic skin injury.
The preparation method of the acellular human fibrin hydrogel comprises the following steps:
step 1: preparing fibrinogen and thrombin into a fibrinogen solution and a thrombin solution respectively, wherein the concentration of the fibrinogen solution is 27mg/mL, and the concentration of the thrombin solution is 5I U/mL;
step 2: sequentially adding calcium chloride and dental pulp stem cell solution into the thrombin solution to obtain thrombin cell suspension, wherein the concentration of the calcium chloride in the thrombin cell suspension is 0mmol/L, and the concentration of the dental pulp stem cells is 0/mL; (i.e., step 2 is not performed, and the thrombin solution in this comparative example is thrombin cell suspension)
Step 3: mixing the fibrinogen solution with the thrombin cell suspension according to the volume ratio of 1:1, incubating for 1h at 37 ℃, and culturing for 12h in a culture medium containing 10% fetal bovine serum to obtain the cell-free human fibrin hydrogel.
For the cell fibroids (example group) and the acellular human fibrin hydrogels (control group) obtained above (example 1) to (example 4), the ability of the same to heal diabetic wounds was examined, in particular, the ability of dental pulp stem cells to secrete leukocyte inhibitory factors (leukocyte inhibitory factor, LIF) which are thought to maintain stem cell stem property and self-renewal ability, and promote angiogenesis in the presence of mesenchymal stem cells, stem cell factor (SCF ) which can promote keratinocyte c-kit receptor activation, promote wound re-epithelialization, and promote vascular regeneration by increasing recruitment of unique stem cell populations present in the skin to some extent, hepatocyte growth factor (hepatocyte growth factor, HGF) which regulates inflammatory status caused by hyperglycemia, promote vascular regeneration, reduce scar formation, promote wound re-epithelialization) and vascular endothelial growth factor (vascular endothelial growth factor, VEGF which mediates vascular endothelial cell proliferation and migration to promote vascular neogenesis, and thus promote vascular healing, are thought to exert the most intense results as shown in the current examination of the results. As a result, it was found that the concentration of cellular fibrinolytic LIF in the example group was significantly higher than that in the control group, wherein the secretion amount of example 2 was significantly higher than that of the other examples; the concentration of secreted SCF was significantly higher in the example group than in the control group, with the secreted amounts of example 2 significantly higher than in the other examples, without significant differences in secreted amounts of example 1 and example 3SCF, with the secreted amounts of example 3SCF significantly higher than in example 4; the concentration of HGF secreted by the example group was significantly higher than that of the control group, with the secretion amount of example 1 significantly higher than that of the other examples, with a significant difference in HGF concentration between example 2 and example 3, and no significant difference in HGF concentration between example 3 and example 4; the concentration of VEGF-A secreted by the example group was significantly higher than that of the control group, wherein the amount secreted by example 1 was significantly higher than that of the other examples, and there was no significant difference in VEGF-A concentration between examples 2,3 and 4. The result shows that the cell fiber glue can obviously promote the dental pulp stem cells to secrete the growth factor LIF, SCF, HGF, VEGF-A; the secretion of LIF, SCF is significantly affected by the formulation and is not consistent with the cell mass; the secretion amount of HGF and VEGF-A is greatly affected by the cell concentration, and the higher the cell concentration is, the higher the concentration of the secreted growth factors HGF and VEGF-A is.
A full-thickness skin wound model was constructed using SPF-grade db/d diabetic mice, and healing of skin wounds of each group was observed by performing no treatment (blank group), treatment with acellular human fibrin hydrogel (control group), and treatment with the cell fibers of the present invention (example group, example 1).
SPF grade db/db diabetic mice (8 weeks old, weight 35-40g, male) were used for intraperitoneal injection anesthesia of diabetic mice with 1% pentobarbital sodium, and after satisfactory anesthesia, the prone position was fixed on the console, and the back skin was sterilized three times with an Aneriodo disinfectant. After the aseptic hole towel, a sterilized puncher is used for punching holes (with the diameter of 8 mm) on a position which is 1cm away from the db/db diabetic mice, and a full-layer skin wound model with the diameter of 8mm which is symmetrical left and right from the back of the diabetic mice to the midline is manufactured.
The same diabetic mice were treated with acellular human fibrin hydrogel on one side (control group), the opposite side wound was not treated (blank group), the double side wound was covered with a 3M transparent dressing, the dressing was changed once on day 2, and the wound healing condition of the skin of each diabetic mouse was recorded by photographing with a digital camera on days 0, 2, 7 and 12 after the operation, as shown in fig. 2, and fig. 3 is a histogram of the healing rate of each group of wounds.
The same diabetic mice were treated with the cell fibers of the present invention on one side of the wound (example group, example 1) and not on the opposite side (blank group), the double-sided wound was covered with a 3M transparent dressing, the dressing was changed once on day 2, and the wound healing of the skin of each diabetic mouse was recorded by photographing with a digital camera on days 0, 2, 7, 12 after the operation, as shown in fig. 4, and fig. 5 is a bar chart of the healing rate of each group of wounds.
The results show that the cell-free fibrin hydrogel is used for treating the full-layer wound surface of the diabetic mice, and the results show that the wound healing rate is not remarkably different from that of the untreated control, and the cell-free fibrin hydrogel has no remarkable treatment effect on the full-layer wound surface of the diabetic mice. The cell fiber gel provided by the invention can obviously promote the repair of diabetic skin injury, increase the wound healing speed, and the wound healing rate of the example group is obviously higher than that of the control group on the treatment day 2, the treatment day 7 and the treatment day 12, which indicates that the cell fiber gel group can obviously promote the healing of diabetic wounds. Specifically, for example, on day 2, the healing efficiency can reach 5.8%; on day 7, the healing efficiency can reach 47.2%; on day 12, the healing efficiency can reach 74.9%. In the control group, the wound healing efficiency is only 3% on the 2 nd day; day 7 was only 22.8%; only 58.5% on day 12.
Observing the skin histochemical staining structures of the blank cell gel group, the example group (taking example 1 as an example) and the normal group (uninjured diabetic mice) respectively, the HE staining and CK14 fluorescent staining results (shown in fig. 6 and 7) show that the wound treated by the cell gel presents a complete epidermis-dermis-subcutaneous tissue structure and a new hair follicle appears; the Masson staining results (shown in fig. 8) showed that the wound treated with cell-fibrillar gel had more collagen tissue, similar to normal skin.
In the analysis of the results, the data comparison among the multiple groups is analyzed by a single factor method, and the data analysis of the paired design is performed by paired t test. p < 0.05 was considered a significant difference.
The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention.
Claims (10)
1. The cell fiber gel for promoting the repair of the skin injury of the diabetes is characterized by comprising the following raw materials:
fibrinogen solution, wherein the concentration of fibrinogen is 23-30 mg/ml;
a thrombin cell suspension comprising dental pulp stem cells, thrombin and calcium chloride, wherein the dental pulp stem cells have a concentration of 5 x 10 4 ～1×10 7 The concentration of thrombin is less than 45IU/mL, and the concentration of calcium chloride is 0-5 mmol/L;
wherein the volume ratio of the fibrinogen solution to the thrombin cell suspension is 1:1.
2. The cell paste of claim 1, wherein the dental pulp stem cells are human dental pulp stem cells.
3. The cell gel according to claim 1, wherein the method for preparing fibrinogen comprises the steps of:
s101: adding the S/D mother solution into the Cohn I solution, and stirring to obtain an inactivated Cohn I solution;
s102: precipitating the inactivated Cohn I solution obtained in the step S101 by using low-temperature ethanol, and centrifuging to obtain fibrinogen precipitate;
s103: and (3) washing the fibrinogen precipitate obtained in the step S102 by using a washing liquid, and packaging.
4. The cell gel according to claim 3, wherein,
the step S101 includes the steps of:
s101-1: dissolving frozen Cohn I to obtain Cohn I solution;
s101-2: carrying out virus inactivation by adopting S/D, adding the S/D mother liquor into the Cohn I solution, wherein the volume ratio of the S/D mother liquor to the Cohn I solution is 1:9, and stirring for 6 hours at the temperature of 24+/-1 ℃ to obtain an inactivated Cohn I solution;
in the step S102, firstly cooling the inactivated Cohn I solution obtained in the step S101-2 to below 0 ℃, then dropwise adding a 50% ethanol solution precooled at the temperature of minus 20 ℃ while stirring until the final concentration of the dropwise adding ethanol is 8%, continuing stirring for 30min after the dropwise adding is finished, standing for 1h, centrifuging at the temperature of minus 2 ℃ for 15min, discarding the supernatant, and collecting fibrinogen precipitate;
the step S103 includes the steps of:
s103-1: the fibrinogen precipitate obtained in the step S102 and the washing liquid are stirred and dissolved at the temperature of 27 ℃ according to the mass ratio of 1:9, and then the step S102 is repeated for one time, and the washing is carried out twice;
s103-2: and (3) stirring and dissolving the fibrinogen precipitate obtained in the step S103-1 and the fibrinogen solution according to the mass ratio of 1:4 at the temperature of 27 ℃ for packaging.
5. The cell gel according to claim 1, wherein the method for preparing thrombin comprises the steps of:
s201: obtaining a plasma-adsorbed gel;
s202: washing and eluting the gel for adsorbing the plasma obtained in the step S201, and filtering and concentrating the eluent;
s203: inactivating viruses by adopting an S/D method;
s204: prothrombin activation;
s205: gel adsorption and purification;
s206: ultrafiltering, concentrating, and packaging;
s207: and inactivating viruses by a dry heat method.
6. The cell gel according to claim 5, wherein in the step S201, after the gel is swelled, the gel is equilibrated with an equilibration solution and added into the cryoprecipitated plasma, and the gel is obtained by stirring and adsorbing the cryoprecipitated plasma at 2-15 ℃ for 30-60 min;
in the step S202, gel adsorbing plasma is washed by using balance liquid and washing liquid in sequence, then eluting is performed by using eluent, and the eluent is collected, filtered and concentrated by ultrafiltration until the protein content is 20+/-10 g/L;
in the step S203, the pH of the product is regulated to 7.00+/-0.20, in the stirring process, S/D solution is added to ensure that the final concentration of the detergent in the product is 1 percent and the final concentration of the organic solvent is 0.3 percent, then the pH of the product is regulated to 7.00+/-0.20 again, and the mixture is stirred and inactivated for 6 hours at the temperature of 24.0+/-2 ℃;
in the step S204, after virus inactivation is finished, the product conductance is regulated to 6-16 ms/cm, 1+/-0.1 mol/L calcium chloride solution is added to enable the final concentration to reach 7mmol/L, the temperature is raised to 37 ℃, the product temperature is kept at 36-38 ℃ for stirring reaction for 1-3 h, then the temperature is reduced to 24 ℃, and the product temperature is kept at 24.0-26.0 ℃ for stirring reaction for 24-30 h;
the step S205 includes the following steps:
s205-1: the gel is firstly washed by 2.0mol/L sodium chloride solution, 1.0 mol/L sodium hydroxide solution and 0.1mol/L sodium chloride solution in sequence, and then is balanced by a balancing solution, and then can be put on a column;
s205-2: adjusting the pH of the product to 6.4-6.6, filtering by a microporous filter membrane with the thickness of 0.22 mu m, loading to a column for adsorption, and controlling the loading amount to be less than 10 times of the volume of the gel column bed;
s205-3: after the sample loading is finished, washing gel by using balance liquid, washing the gel by using washing liquid, eluting the gel by using eluent, collecting the eluent according to a chromatographic chart, and adding 60ml of 1+/-0.1 mol/L calcium chloride solution, 2g of human serum albumin, 5g of sucrose and 3g of glycine into each liter of product after measuring the volume;
the step S206 includes the steps of:
s206-1: dialyzing the product prepared in the step S205-3 in a cold storage at 4 ℃ overnight, sampling to determine the titer of thrombin, diluting the product with dialysate to make the titer of thrombin less than 550IU/ml, supplementing human serum albumin to 5g/L, and controlling the pH of the product to be 6.3-7.6;
s206-2: packaging the above products, freeze-drying at-60deg.C under vacuum degree of 0.001mbar for about 48 hr;
in the step S207, the freeze-dried human thrombin sample obtained in the step S206-2 is placed in a water bath box, a thermometer is placed at multiple points, timing is started when the temperature reaches 99 ℃, the temperature is recorded every 5min, the water bath temperature is controlled within the range of 99-100 ℃ in the inactivation process, and heating is kept for 30min.
7. A method for preparing a cell fiber gel for promoting repair of diabetic skin injury, which can be used for preparing the cell fiber gel according to any one of claims 1 to 6, comprising the following steps:
step 1: respectively preparing fibrinogen and thrombin into a fibrinogen solution and a thrombin solution, wherein the concentration of the fibrinogen solution is 23-30 mg/mL, and the concentration of the thrombin solution is less than 45U/mL;
step 2: sequentially adding calcium chloride and dental pulp stem cell solution into the thrombin solution to obtain thrombin cell suspension, wherein the concentration of calcium chloride in the thrombin cell suspension is 0-5 mmol/L, and the concentration of dental pulp stem cells is 5 multiplied by 10 4 ～1×10 7 individual/mL;
step 3: mixing the fibrinogen solution with the thrombin cell suspension according to the volume ratio of 1:1, incubating for 1h at 37 ℃, and culturing for 12h in a culture medium containing 10% fetal bovine serum to obtain the cell fiber gel for promoting the repair of the diabetic skin injury.
8. The method according to claim 7, wherein in step 1, the fibrinogen is pretreated before being prepared into a solution, and the pretreatment process is as follows: the fibrinogen is pre-frozen overnight at the temperature of-70 ℃, then placed in a freeze dryer for vacuum freeze drying, the working condition is that a cold trap is stable at-60 ℃, the vacuum degree is 0.001mbar, and the freeze drying time is 72 hours.
9. The method according to claim 8, wherein in step 1, the thrombin is pretreated before being formulated into a solution, and the pretreatment process is as follows: the thrombin solution was lyophilized at 60℃under a vacuum of 0.001mbar for 48h.
10. The method according to claim 7, wherein the solvent of all the solutions is distilled water having a pH of 7 and subjected to autoclaving;
in the step 3, the thickness of the single fibrin hydrogel is less than or equal to 2mm.
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