Disclosure of Invention
The invention aims to provide a fat stem cell composition for wound repair, a preparation method and application thereof, which have the effects of better implantation into damaged tissues, release of nutrients, promotion of neovascularization, regulation of oxidative stress and induction of anti-inflammatory reaction, and are beneficial to wound repair, and can be converted into gel phase, stabilize components and release in a sustained and controlled manner at the skin epidermis temperature of 35-37 ℃.
The technical scheme of the invention is realized as follows:
The invention provides a preparation method of a fat stem cell composition for wound repair, which comprises the steps of adding fetal calf serum, dexamethasone, penicillin, streptomycin, collagen, magnetic particles and P substances into a DMEM culture medium, preparing the culture medium, culturing fat mesenchymal stem cells by a low-frequency magnetic field, separating to obtain a stem cell solution, embedding to obtain stem cell microspheres, freeze-drying supernatant to obtain paracrine culture substances, and uniformly stirring and mixing the paracrine culture substances with the stem cell microspheres, hydroxypropyl methylcellulose, carboxymethyl chitosan, hyaluronic acid, glycerol and the stem cell microspheres to obtain the fat stem cell composition for wound repair.
As a further improvement of the invention, the method comprises the following steps:
S1, preparing a culture medium, namely adding fetal bovine serum, dexamethasone, penicillin, streptomycin, collagen, magnetic particles and substances P into the DMEM culture medium to prepare the culture medium;
S2, culturing adipose-derived mesenchymal stem cells, namely resuscitating the adipose-derived mesenchymal stem cells, adding the resuscitated adipose-derived mesenchymal stem cells into a culture medium, culturing the cells by a low-frequency magnetic field, separating magnetic substances by a magnet after the cells are grown to 80-90% fusion by adherence, centrifuging residues, collecting supernatant, flushing solid by using D-Hank' S liquid, digesting, resuspending the solid in a DMEM culture medium, and freezing to prepare a stem cell solution;
s3, treating a culture solution, namely freeze-drying the supernatant obtained in the step S2 to obtain paracrine culture substances;
S4, preparing silk fibroin peptide scaffold particles, namely dissolving silk fibroin peptide and lecithin in water to obtain a solution, dripping the solution into liquid paraffin, emulsifying, adding EDC and NHS mixed solution, stirring, solidifying, centrifuging, washing and drying to obtain the silk fibroin peptide scaffold particles;
S5, preparing stem cell microspheres, namely resuscitating a stem cell solution, adding silk fibroin peptide scaffold particles, stirring and adsorbing, adding sodium alginate and lecithin, stirring and uniformly mixing, dripping into liquid paraffin, emulsifying, dripping calcium chloride solution, curing at normal temperature, centrifuging, washing and drying to obtain the stem cell microspheres;
S6, preparing temperature-sensitive hydrogel, namely dissolving hydroxypropyl methylcellulose in hot water, cooling to room temperature, adding carboxymethyl chitosan, hyaluronic acid and glycerol, stirring and mixing uniformly, placing at 4 ℃, adding stem cell microspheres and paracrine culture substances, stirring and mixing uniformly, and obtaining the adipose-derived stem cell composition for wound repair.
As a further improvement of the invention, the content of fetal bovine serum in the culture medium in the step S1 is 9-11wt%, the content of dexamethasone is 5-15nmol/L, the content of penicillin is 100-120U/mL, the content of streptomycin is 100-120 mug/mL, the content of collagen is 70-100 mug/mL, the content of magnetic particles is 100-200 mug/mL, the content of P substance is 3-5 mug/mL, and the magnetic particles are micron-sized ferroferric oxide.
As a further improvement of the invention, the low-frequency magnetic field in the step S2 is used for culturing for 1-2 weeks, the culturing condition is 36-38 ℃, the concentration of CO2 is 3-6v/v%, the frequency of the low-frequency magnetic field is 40-50Hz, the stimulation time is 30-40min/d, and the digestion solution is PBS buffer solution containing 1-2wt% of trypsin and 0.5-1wt% of alkaline protease and having the pH of 7.5-7.7.
As a further improvement of the invention, the mass ratio of the silk fibroin peptide, lecithin, EDC and NHS in the step S4 is 15-20:0.5-1:0.1-0.2:0.15-0.25.
As a further improvement of the invention, the mass ratio of the stem cell solution, the silk fibroin peptide scaffold particles, the sodium alginate and the lecithin in the step S5 is 100:12-15:17-22:1-2.
As a further improvement of the invention, the temperature of the hot water in the step S6 is 70-80 ℃, and the mass ratio of the hydroxypropyl methylcellulose, the carboxymethyl chitosan, the hyaluronic acid, the glycerol, the stem cell microspheres and the paracrine culture substance is 10-12:5-7:3-4:2-4:7-10:4-6.
As a further improvement of the invention, the method specifically comprises the following steps:
S1, preparing a culture medium, namely adding fetal bovine serum, dexamethasone, penicillin, streptomycin, collagen, micron-sized ferroferric oxide and P substances into the DMEM culture medium to prepare the culture medium;
the content of fetal bovine serum in the culture medium is 9-11wt%, the content of dexamethasone is 5-15nmol/L, the content of penicillin is 100-120U/mL, the content of streptomycin is 100-120 mug/mL, the content of collagen is 70-100 mug/mL, the content of micron-sized ferroferric oxide is 100-200 mug/mL, and the content of substance P is 3-5 mug/mL;
S2, culturing adipose-derived mesenchymal stem cells, namely resuscitating the adipose-derived mesenchymal stem cells, adding the resuscitated adipose-derived mesenchymal stem cells into a culture medium, performing low-frequency magnetic field culture for 1-2 weeks at 36-38 ℃ with the concentration of CO2 being 3-6v/v%, wherein the frequency of the low-frequency magnetic field is 40-50Hz, the stimulation time is 30-40min/D, separating magnetic substances by a magnet after the cells are grown to 80-90% of the cells are fused, centrifuging residues, collecting supernatant, washing solids by using D-Hank' S liquid, digesting, re-suspending the solids in the DMEM culture medium, and performing freeze-preservation to obtain a stem cell solution;
the digestion solution is PBS buffer solution containing 1-2wt% of trypsin and 0.5-1wt% of alkaline protease and having a pH of 7.5-7.7;
s3, treating a culture solution, namely freeze-drying the supernatant obtained in the step S2 to obtain paracrine culture substances;
S4, preparing silk fibroin peptide scaffold particles, namely dissolving 15-20 parts by weight of silk fibroin peptide and 0.5-1 part by weight of lecithin in water to obtain a solution, dropwise adding the solution into liquid paraffin, emulsifying, adding 20mL of mixed solution containing 0.1-0.2 part by weight of EDC and 0.15-0.25 part by weight of NHS, stirring, solidifying, centrifuging, washing and drying to obtain the silk fibroin peptide scaffold particles;
S5, preparing stem cell microspheres, namely resuscitating 100 parts by weight of stem cell solution, adding 12-15 parts by weight of silk fibroin peptide scaffold particles, stirring and adsorbing, adding 17-22 parts by weight of sodium alginate and 1-2 parts by weight of lecithin, stirring and mixing uniformly, dripping into liquid paraffin, emulsifying, dripping calcium chloride solution, solidifying at normal temperature, centrifuging, washing and drying to prepare the stem cell microspheres;
S6, preparing temperature-sensitive hydrogel, namely dissolving 10-12 parts by weight of hydroxypropyl methylcellulose in 70-80 ℃ hot water, cooling to room temperature, adding 5-7 parts by weight of carboxymethyl chitosan, 3-4 parts by weight of hyaluronic acid and 2-4 parts by weight of glycerin, stirring and mixing uniformly, placing at 4 ℃, adding 7-10 parts by weight of stem cell microspheres and 4-6 parts by weight of paracrine culture substances, stirring and mixing uniformly, and obtaining the adipose-derived stem cell composition for wound repair.
The invention further provides the adipose-derived stem cell composition for wound repair, which is prepared by the preparation method.
The invention further provides application of the adipose-derived stem cell composition for repairing wounds in preparation of medicines for repairing skin wounds.
The invention has the following beneficial effects:
Wound healing is a highly complex biological process consisting of three overlapping stages, inflammation, proliferation and remodeling. Any one or more stages of injury may result in a repair failure. Adipose-derived mesenchymal stem cells can be implanted into damaged tissues, release nutrients, promote neovascularization, regulate oxidative stress and induce anti-inflammatory responses. The epidermal cells and the fibroblasts play an important role in the skin wound repair process, participate in a plurality of physiological processes such as wound repair activation, proliferation and re-epithelialization, wound contraction, tissue reconstruction, extracellular matrix deposition and the like, and the adipose mesenchymal stem cells can be directly differentiated into cell lines required by skin wound repair such as an epidermal cell line, a fibroblast line and an endothelial cell line under the influence of local microenvironment, and simultaneously secrete growth factors to promote proliferation and migration of the epidermal cells, the fibroblasts and the endothelial cells so as to promote skin wound healing.
The collagen is added into the culture medium, is an important raw material substance for repairing tissues after the skin is damaged, provides a raw material for skin repair, can promote proliferation and differentiation of stem cells, promote paracrine function and improve secretion of exosomes.
The micro-scale ferroferric oxide is a magnetic substance, and in the low-frequency magnetic field culture process, a micro-magnetic field is formed by attaching the micro-magnetic field to the surface of stem cells in response to a magnetic field, so that the structure of cell membranes and cell membrane related enzymes are influenced to increase the permeability of small molecular substances, thereby changing the cell behavior by means of the action of second messengers, and simultaneously, the opening of a voltage-gated calcium channel is controlled to cause the inflow of calcium ions, the increase of the concentration of calcium ions in the cells and the growth and development functions of the cells are promoted;
Substance P is a highly conserved neuropeptide, called "injury inducing factor" by some researchers, and has the ability to regulate inflammation, recruit stem cells, promote cell proliferation and migration, and promote vascularization, and at the same time, can enhance the paracrine function of stem cells, thus playing a role in synergistically promoting regeneration.
Silk fibroin peptide is a protein peptide with good biocompatibility, and has good mechanical strength, glutaraldehyde is a common chemical crosslinking agent, but glutaraldehyde crosslinking products are usually toxic substances released by biodegradation, so that the repair of skin wound surfaces is not facilitated. EDC and NHS can help the carboxyl and amino in the silk fibroin peptide molecule to form an amide bond, but the EDC and NHS do not become a part of actual crosslinking, and the EDC and NHS have the characteristics of no toxicity and good biocompatibility, so that the safety and biocompatibility of the prepared product are improved. Through the hydrogen bond adsorption effect of the glycoprotein on the surface of the protein peptide and the stem cell, the stem cell can be adsorbed and fixed on the silk fibroin peptide scaffold particles, and then coated in the sodium alginate microsphere, so as to replace water molecules to form a hydration bond with hydrophilic groups on cell membranes and proteins, avoid the change of the cell membrane and protein molecular structure, keep the stability of the structure and the function of the cell membrane and the protein molecular structure, ensure that the stem cell can be stored for a long time, improve the storage stability of the stem cell and also improve the storage stability of exosomes.
Hydroxypropyl methyl cellulose is a cellulose-derived temperature-sensitive hydrophilic polymer, has the advantages of no toxicity, low cost, high swelling property, surface activity and the like, and hydrogel prepared by mixing the hydroxypropyl methyl cellulose with glycerol and hyaluronic acid has temperature-sensitive property and can be subjected to sol-gel phase transition according to the change of environmental temperature. The paracrine culture substance contains collagen, can form temperature-sensitive hydrogel with chitosan, and can shorten the gel time and improve the crosslinking degree, mechanical strength and biocompatibility of the hydrogel by introducing the collagen. Through the mixing of two different hydrogel systems, the phase transition temperature of the hydrogel can be adjusted under the interaction, so that the hydrogel can be converted into a gel phase at the temperature of 35-37 ℃ of the epidermis of the skin, and is a solution phase below the temperature, thereby being convenient for coating and forming gel on the surface of a wound surface, slowly releasing the medicine, isolating oxygen and promoting the rapid healing and recovery of the wound surface.
The fat stem cell composition for wound repair, which is prepared by the invention, has the effects of better implanting damaged tissues, releasing nutrient substances, promoting neovascularization, regulating oxidative stress and inducing anti-inflammatory reaction, is beneficial to wound repair, can be converted into gel phase at the skin epidermis temperature of 35-37 ℃, and is stable in component and released in a sustained and controlled manner.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Collagen, which is a collagen tripeptide with a content of >99% and is available from the company of the Biotechnology of saline rain, inc., 280-300 Da. Micron-sized ferroferric oxide, 200 mesh, purchased from norvex welding materials, inc. Substance P, CAS number 33507-63-0, content >98%, brand Widely, available from Hubei Widli chemical Co., ltd. Trypsin, 20U/g, alkaline protease, 20U/g, purchased from Xia Cheng (Beijing) enzyme Biotechnology development Co., ltd.
NHS, N-hydroxysuccinimide, EDC,1- (3-dimethylaminopropyl) -3-ethylcarbodiimide.
Example 1
The embodiment provides a preparation method of a fat stem cell composition for wound repair, which specifically comprises the following steps:
S1, preparing a culture medium, namely adding fetal bovine serum, dexamethasone, penicillin, streptomycin, collagen, micron-sized ferroferric oxide and P substances into the DMEM culture medium to prepare the culture medium;
The content of fetal bovine serum in the culture medium is 9wt%, the content of dexamethasone is 5nmol/L, the content of penicillin is 100U/mL, the content of streptomycin is 100 mug/mL, the content of collagen is 70 mug/mL, the content of micron-sized ferroferric oxide is 100 mug/mL, and the content of substance P is 3 mug/mL;
S2, culturing adipose-derived mesenchymal stem cells, namely resuscitating the adipose-derived mesenchymal stem cells, adding the resuscitated adipose-derived mesenchymal stem cells into a culture medium, culturing for 1 week at 36 ℃ under a low-frequency magnetic field with the CO2 concentration of 3v/v%, wherein the frequency of the low-frequency magnetic field is 40Hz, the stimulation time is 30min/D, separating magnetic substances by a magnet after the cells are grown to 80-90% of fusion, centrifuging residues, collecting supernatant, flushing solids by using D-Hank' S liquid, digesting the digested solids, re-suspending the digested solids in the DMEM culture medium, and freezing to obtain a stem cell solution;
The digestion solution is PBS buffer solution containing 1wt% of trypsin and 0.5wt% of alkaline protease and having pH of 7.5;
s3, treating a culture solution, namely freeze-drying the supernatant obtained in the step S2 to obtain paracrine culture substances;
S4, preparing silk fibroin peptide scaffold particles, namely dissolving 15g of silk fibroin peptide and 0.5g of lecithin in 300mL of water to obtain a solution, dropwise adding the solution into 500mL of liquid paraffin, emulsifying for 15min at 7000r/min, adding 20mL of mixed solution containing 0.1g of EDC and 0.15g of NHS, stirring and solidifying for 2h, centrifuging, washing and drying to obtain the silk fibroin peptide scaffold particles;
S5, preparing stem cell microspheres, namely resuscitating 100g of stem cell solution, adding 12g of silk fibroin peptide scaffold particles, stirring and adsorbing for 20min, adding 17g of sodium alginate and 1g of lecithin, stirring and mixing for 10min, dropwise adding into 300mL of liquid paraffin, emulsifying for 15min at 7000r/min, dropwise adding 20mL of 5wt% calcium chloride solution, solidifying for 30min at normal temperature, centrifuging, washing, and drying to obtain the stem cell microspheres;
S6, preparing temperature-sensitive hydrogel, namely dissolving 10g of hydroxypropyl methylcellulose into 500mL of 70 ℃ hot water, cooling to room temperature, adding 5g of carboxymethyl chitosan, 3g of hyaluronic acid and 2g of glycerol, stirring and mixing for 15min, and adding 7g of stem cell microspheres and 4g of paracrine culture substance when the temperature is 4 ℃, stirring and mixing for 15min to obtain the adipose-derived stem cell composition for wound repair.
Example 2
The embodiment provides a preparation method of a fat stem cell composition for wound repair, which specifically comprises the following steps:
S1, preparing a culture medium, namely adding fetal bovine serum, dexamethasone, penicillin, streptomycin, collagen, micron-sized ferroferric oxide and P substances into the DMEM culture medium to prepare the culture medium;
the content of fetal bovine serum in the culture medium is 11wt%, the content of dexamethasone is 15nmol/L, the content of penicillin is 120U/mL, the content of streptomycin is 120 mug/mL, the content of collagen is 100 mug/mL, the content of micron-sized ferroferric oxide is 200 mug/mL, and the content of substance P is 5 mug/mL;
s2, culturing adipose-derived mesenchymal stem cells, namely resuscitating the adipose-derived mesenchymal stem cells, adding the resuscitated adipose-derived mesenchymal stem cells into a culture medium, culturing for 2 weeks at 38 ℃ under a low-frequency magnetic field with the CO2 concentration of 6v/v%, wherein the frequency of the low-frequency magnetic field is 50Hz, the stimulation time is 40min/D, separating magnetic substances by a magnet after the cells are grown to 80-90% of fusion, centrifuging residues, collecting supernatant, flushing solids by using D-Hank' S liquid, digesting the digested solids, re-suspending the digested solids in the DMEM culture medium, and freezing to obtain a stem cell solution;
The digestion solution is PBS buffer solution containing 2wt% of trypsin and 1wt% of alkaline protease and having a pH of 7.7;
s3, treating a culture solution, namely freeze-drying the supernatant obtained in the step S2 to obtain paracrine culture substances;
S4, preparing silk fibroin peptide scaffold particles, namely dissolving 20g of silk fibroin peptide and 1g of lecithin in 300mL of water to obtain a solution, dropwise adding the solution into 500mL of liquid paraffin, emulsifying for 15min at 7000r/min, adding 20mL of mixed solution containing 0.2g of EDC and 0.25g of NHS, stirring and solidifying for 2h, centrifuging, washing and drying to obtain the silk fibroin peptide scaffold particles;
S5, preparing stem cell microspheres, namely resuscitating 100g of stem cell solution, adding 15g of silk fibroin peptide scaffold particles, stirring and adsorbing for 20min, adding 22g of sodium alginate and 2g of lecithin, stirring and mixing for 10min, dropwise adding into 300mL of liquid paraffin, emulsifying for 15min at 7000r/min, dropwise adding 20mL of 5wt% calcium chloride solution, solidifying for 30min at normal temperature, centrifuging, washing, and drying to obtain the stem cell microspheres;
S6, preparing temperature-sensitive hydrogel, namely dissolving 12g of hydroxypropyl methylcellulose in 500mL of 80 ℃ hot water, cooling to room temperature, adding 7g of carboxymethyl chitosan, 4g of hyaluronic acid and 4g of glycerol, stirring and mixing for 15min, and adding 10g of stem cell microspheres and 6g of paracrine culture substance when the temperature is set at 4 ℃, stirring and mixing for 15min to obtain the adipose-derived stem cell composition for wound repair.
Example 3
The embodiment provides a preparation method of a fat stem cell composition for wound repair, which specifically comprises the following steps:
S1, preparing a culture medium, namely adding fetal bovine serum, dexamethasone, penicillin, streptomycin, collagen, micron-sized ferroferric oxide and P substances into the DMEM culture medium to prepare the culture medium;
The content of fetal bovine serum in the culture medium is 10wt%, the content of dexamethasone is 10nmol/L, the content of penicillin is 110U/mL, the content of streptomycin is 110 mug/mL, the content of collagen is 85 mug/mL, the content of micron-sized ferroferric oxide is 150 mug/mL, and the content of substance P is 4 mug/mL;
S2, culturing adipose-derived mesenchymal stem cells, namely resuscitating the adipose-derived mesenchymal stem cells, adding the resuscitated adipose-derived mesenchymal stem cells into a culture medium, culturing for 2 weeks at 37 ℃ under a low-frequency magnetic field with the CO2 v/v% and the frequency of 45Hz, wherein the stimulation time is 35min/D, separating magnetic substances by a magnet after the cells are grown to 80-90% of fusion, centrifuging residues, collecting supernatant, flushing solids by using D-Hank' S liquid, digesting the digested solids, re-suspending the digested solids in the DMEM culture medium, and freezing to obtain a stem cell solution;
the digestion solution is PBS buffer solution containing 1.5wt% trypsin and 0.7wt% alkaline protease and having pH of 7.6;
s3, treating a culture solution, namely freeze-drying the supernatant obtained in the step S2 to obtain paracrine culture substances;
S4, preparing silk fibroin peptide scaffold particles, namely dissolving 17g of silk fibroin peptide and 0.7g of lecithin in 300mL of water to obtain a solution, dropwise adding the solution into 500mL of liquid paraffin, emulsifying for 15min at 7000r/min, adding 20mL of mixed solution containing 0.15g of EDC and 0.2g of NHS, stirring and solidifying for 2h, centrifuging, washing and drying to obtain the silk fibroin peptide scaffold particles;
S5, preparing stem cell microspheres, namely resuscitating 100g of stem cell solution, adding 13g of silk fibroin peptide scaffold particles, stirring and adsorbing for 20min, adding 20g of sodium alginate and 1.5g of lecithin, stirring and mixing for 10min, dropwise adding into 300mL of liquid paraffin, emulsifying for 15min at 7000r/min, dropwise adding 20mL of 5wt% calcium chloride solution, solidifying for 30min at normal temperature, centrifuging, washing and drying to obtain the stem cell microspheres;
S6, preparing temperature-sensitive hydrogel, namely dissolving 11g of hydroxypropyl methylcellulose in 500mL of 75 ℃ hot water, cooling to room temperature, adding 6g of carboxymethyl chitosan, 3.5g of hyaluronic acid and 3g of glycerol, stirring and mixing for 15min, adding 8g of stem cell microspheres and 5g of paracrine culture substance when the temperature is set at 4 ℃, and stirring and mixing for 15min to obtain the adipose-derived stem cell composition for wound repair.
Comparative example 1
The difference from example 3 is that no collagen was added in step S1.
The method comprises the following steps:
s1, preparing a culture medium, namely adding fetal calf serum, dexamethasone, penicillin, streptomycin, micron-sized ferroferric oxide and P substances into the DMEM culture medium to prepare the culture medium;
The content of fetal bovine serum in the culture medium is 10wt%, the content of dexamethasone is 10nmol/L, the content of penicillin is 110U/mL, the content of streptomycin is 110 mug/mL, the content of micron-sized ferroferric oxide is 150 mug/mL, and the content of substance P is 4 mug/mL.
Comparative example 2
In comparison with example 3, the difference is that no micro-sized ferroferric oxide was added in step S1.
The method comprises the following steps:
s1, preparing a culture medium, namely adding fetal bovine serum, dexamethasone, penicillin, streptomycin, collagen and substances P into the DMEM culture medium to prepare the culture medium;
the content of fetal bovine serum in the culture medium is 10wt%, the content of dexamethasone is 10nmol/L, the content of penicillin is 110U/mL, the content of streptomycin is 110 mug/mL, the content of collagen is 85 mug/mL, and the content of substance P is 4 mug/mL.
Comparative example 3
The difference from example 3 is that substance P is not added in step S1.
The method comprises the following steps:
s1, preparing a culture medium, namely adding fetal calf serum, dexamethasone, penicillin, streptomycin, collagen and micron-sized ferroferric oxide into the DMEM culture medium to prepare the culture medium;
The content of fetal bovine serum in the culture medium is 10wt%, the content of dexamethasone is 10nmol/L, the content of penicillin is 110U/mL, the content of streptomycin is 110 mug/mL, the content of collagen is 85 mug/mL, and the content of micron-sized ferroferric oxide is 150 mug/mL.
Comparative example 4
In comparison with example 3, the difference is that the low-frequency magnetic field culture is not performed in step S2.
The method comprises the following steps:
S2, culturing the adipose-derived mesenchymal stem cells, namely resuscitating the adipose-derived mesenchymal stem cells, adding the resuscitated adipose-derived mesenchymal stem cells into a culture medium, culturing for 2 weeks at 37 ℃ with the concentration of CO2 v/v%, separating magnetic substances by a magnet after the cells are grown to 80-90% by adherence, centrifuging residues, collecting supernatant, flushing solids by using D-Hank' S liquid, digesting the digested liquid, re-suspending the digested solid in a DMEM culture medium, and freezing to obtain a stem cell solution;
The digestion solution was a PBS buffer at pH 7.6 containing 1.5wt% trypsin, 0.7wt% alkaline protease.
Comparative example 5
In comparison with example 3, the difference is that step S4 is not performed.
The method comprises the following steps:
S1, preparing a culture medium, namely adding fetal bovine serum, dexamethasone, penicillin, streptomycin, collagen, micron-sized ferroferric oxide and P substances into the DMEM culture medium to prepare the culture medium;
The content of fetal bovine serum in the culture medium is 10wt%, the content of dexamethasone is 10nmol/L, the content of penicillin is 110U/mL, the content of streptomycin is 110 mug/mL, the content of collagen is 85 mug/mL, the content of micron-sized ferroferric oxide is 150 mug/mL, and the content of substance P is 4 mug/mL;
S2, culturing adipose-derived mesenchymal stem cells, namely resuscitating the adipose-derived mesenchymal stem cells, adding the resuscitated adipose-derived mesenchymal stem cells into a culture medium, culturing for 2 weeks at 37 ℃ under a low-frequency magnetic field with the CO2 v/v% and the frequency of 45Hz, wherein the stimulation time is 35min/D, separating magnetic substances by a magnet after the cells are grown to 80-90% of fusion, centrifuging residues, collecting supernatant, flushing solids by using D-Hank' S liquid, digesting the digested solids, re-suspending the digested solids in the DMEM culture medium, and freezing to obtain a stem cell solution;
the digestion solution is PBS buffer solution containing 1.5wt% trypsin and 0.7wt% alkaline protease and having pH of 7.6;
s3, treating a culture solution, namely freeze-drying the supernatant obtained in the step S2 to obtain paracrine culture substances;
S4, preparing stem cell microspheres, namely resuscitating 100g of stem cell solution, adding 20g of sodium alginate and 1.5g of lecithin, stirring and mixing for 10min, dropwise adding into 300mL of liquid paraffin, emulsifying for 15min at 7000r/min, dropwise adding 20mL of 5wt% calcium chloride solution, solidifying for 30min at normal temperature, centrifuging, washing and drying to obtain the stem cell microspheres;
S5, preparing temperature-sensitive hydrogel, namely dissolving 11g of hydroxypropyl methylcellulose in 500mL of 75 ℃ hot water, cooling to room temperature, adding 6g of carboxymethyl chitosan, 3.5g of hyaluronic acid and 3g of glycerol, stirring and mixing for 15min, adding 8g of stem cell microspheres and 5g of paracrine culture substance when the temperature is set at 4 ℃, and stirring and mixing for 15min to obtain the adipose-derived stem cell composition for wound repair.
Comparative example 6
In comparison with example 3, the difference is that the embedding of step S5 is not performed.
The method comprises the following steps:
S1, preparing a culture medium, namely adding fetal bovine serum, dexamethasone, penicillin, streptomycin, collagen, micron-sized ferroferric oxide and P substances into the DMEM culture medium to prepare the culture medium;
The content of fetal bovine serum in the culture medium is 10wt%, the content of dexamethasone is 10nmol/L, the content of penicillin is 110U/mL, the content of streptomycin is 110 mug/mL, the content of collagen is 85 mug/mL, the content of micron-sized ferroferric oxide is 150 mug/mL, and the content of substance P is 4 mug/mL;
S2, culturing adipose-derived mesenchymal stem cells, namely resuscitating the adipose-derived mesenchymal stem cells, adding the resuscitated adipose-derived mesenchymal stem cells into a culture medium, culturing for 2 weeks at 37 ℃ under a low-frequency magnetic field with the CO2 v/v% and the frequency of 45Hz, wherein the stimulation time is 35min/D, separating magnetic substances by a magnet after the cells are grown to 80-90% of fusion, centrifuging residues, collecting supernatant, flushing solids by using D-Hank' S liquid, digesting the digested solids, re-suspending the digested solids in the DMEM culture medium, and freezing to obtain a stem cell solution;
the digestion solution is PBS buffer solution containing 1.5wt% trypsin and 0.7wt% alkaline protease and having pH of 7.6;
s3, treating a culture solution, namely freeze-drying the supernatant obtained in the step S2 to obtain paracrine culture substances;
S4, preparing silk fibroin peptide scaffold particles, namely dissolving 17g of silk fibroin peptide and 0.7g of lecithin in 300mL of water to obtain a solution, dropwise adding the solution into 500mL of liquid paraffin, emulsifying for 15min at 7000r/min, adding 20mL of mixed solution containing 0.15g of EDC and 0.2g of NHS, stirring and solidifying for 2h, centrifuging, washing and drying to obtain the silk fibroin peptide scaffold particles;
S5, preparing stem cell particles, namely resuscitating 100g of stem cell solution, adding 13g of silk fibroin peptide scaffold particles, and stirring and adsorbing for 20min to prepare the stem cell particles;
S6, preparing temperature-sensitive hydrogel, namely dissolving 11g of hydroxypropyl methylcellulose in 500mL of 75 ℃ hot water, cooling to room temperature, adding 6g of carboxymethyl chitosan, 3.5g of hyaluronic acid and 3g of glycerol, stirring and mixing for 15min, adding 8g of stem cell particles and 5g of paracrine culture substance when the temperature is set at 4 ℃, and stirring and mixing for 15min to obtain the adipose-derived stem cell composition for wound repair.
Comparative example 7
In comparison with example 3, the difference is that step S6 is not performed.
The method comprises the following steps:
S1, preparing a culture medium, namely adding fetal bovine serum, dexamethasone, penicillin, streptomycin, collagen, micron-sized ferroferric oxide and P substances into the DMEM culture medium to prepare the culture medium;
The content of fetal bovine serum in the culture medium is 10wt%, the content of dexamethasone is 10nmol/L, the content of penicillin is 110U/mL, the content of streptomycin is 110 mug/mL, the content of collagen is 85 mug/mL, the content of micron-sized ferroferric oxide is 150 mug/mL, and the content of substance P is 4 mug/mL;
S2, culturing adipose-derived mesenchymal stem cells, namely resuscitating the adipose-derived mesenchymal stem cells, adding the resuscitated adipose-derived mesenchymal stem cells into a culture medium, culturing for 2 weeks at 37 ℃ under a low-frequency magnetic field with the CO2 v/v% and the frequency of 45Hz, wherein the stimulation time is 35min/D, separating magnetic substances by a magnet after the cells are grown to 80-90% of fusion, centrifuging residues, collecting supernatant, flushing solids by using D-Hank' S liquid, digesting the digested solids, re-suspending the digested solids in the DMEM culture medium, and freezing to obtain a stem cell solution;
the digestion solution is PBS buffer solution containing 1.5wt% trypsin and 0.7wt% alkaline protease and having pH of 7.6;
s3, treating a culture solution, namely freeze-drying the supernatant obtained in the step S2 to obtain paracrine culture substances;
S4, preparing silk fibroin peptide scaffold particles, namely dissolving 17g of silk fibroin peptide and 0.7g of lecithin in 300mL of water to obtain a solution, dropwise adding the solution into 500mL of liquid paraffin, emulsifying for 15min at 7000r/min, adding 20mL of mixed solution containing 0.15g of EDC and 0.2g of NHS, stirring and solidifying for 2h, centrifuging, washing and drying to obtain the silk fibroin peptide scaffold particles;
S5, preparing stem cell microspheres, namely resuscitating 100g of stem cell solution, adding 13g of silk fibroin peptide scaffold particles, stirring and adsorbing for 20min, adding 20g of sodium alginate and 1.5g of lecithin, stirring and mixing for 10min, dropwise adding into 300mL of liquid paraffin, emulsifying for 15min at 7000r/min, dropwise adding 20mL of 5wt% calcium chloride solution, solidifying for 30min at normal temperature, centrifuging, washing and drying to obtain the stem cell microspheres;
S6, preparing the adipose-derived stem cell composition for wound repair, namely stirring and mixing 8g of stem cell microspheres and 5g of paracrine culture substance for 15min to obtain the adipose-derived stem cell composition for wound repair.
Comparative example 8
The difference from example 3 is that step S6 is different.
The method comprises the following steps:
S6, preparing temperature-sensitive hydrogel, namely adding 6g of carboxymethyl chitosan and 3.5g of hyaluronic acid into 500mL of water, stirring and mixing for 15min, placing at 4 ℃, adding 8g of stem cell particles and 5g of paracrine culture substance, and stirring and mixing for 15min to obtain the fat stem cell composition for wound repair.
Comparative example 9
The difference from example 3 is that step S6 is different.
The method comprises the following steps:
S6, preparing temperature-sensitive hydrogel, namely dissolving 11g of hydroxypropyl methylcellulose into 500mL of 75 ℃ hot water, cooling to room temperature, adding 3.5g of hyaluronic acid and 3g of glycerol, stirring and mixing for 15min, adding 8g of stem cell particles and 5g of paracrine culture substance when the temperature is 4 ℃, and stirring and mixing for 15min to obtain the adipose-derived stem cell composition for wound repair.
Comparative example 10
In comparison with example 3, the difference is that glutaraldehyde is used instead of EDC and NHS in step S4.
The method comprises the following steps:
S4, preparing silk fibroin peptide scaffold particles, namely dissolving 17g of silk fibroin peptide and 0.7g of lecithin in 300mL of water to obtain a solution, dropwise adding the solution into 500mL of liquid paraffin, emulsifying for 15min at 7000r/min, adding 20mL of aqueous solution containing 0.35g of glutaraldehyde, stirring and solidifying for 2h, centrifuging, washing and drying to obtain the silk fibroin peptide scaffold particles.
Test example 1
The fat stem cell compositions for wound repair prepared in examples 1 to 3 and comparative examples 8 and 9 were taken in glass test tubes, placed in a thermostat water bath, the test tubes were taken out every 10 min, inverted to see whether the hydrogel flowed, if flowing, the temperature was continued to rise at a rate of 1 ℃, and if not, the temperature was defined as the gelation temperature Tgel of the hydrogel. The results are shown in Table 1.
TABLE 1
As is clear from the above table, the adipose-derived stem cell compositions for wound repair prepared in examples 1 to 3 of the present invention have a suitable gelation temperature.
Test example 2
The adipose-derived stem cell compositions for wound repair prepared in examples 1 to 3 and comparative example 10 were added in equal volumes to 10mL of DMEM medium containing 10% fetal bovine serum and 1% penicillin/streptomycin as a sample medium. L929 was seeded at a density of 2X 104/mL into 96-well plates and cultured in a 37℃incubator containing a volume fraction of 5% CO2 for 12h until cells were fully adherent. The culture medium of each group was aspirated, the sample medium was added to each of the experimental groups, the DMEM complete medium was added to the control group, and 6 parallel wells were provided for each group. The plates were removed on day 5, and 10. Mu.L of CCK-solution (CCK-8 kit, dojindo, japan) was added to each well and incubated at 37℃for 2h in the absence of light. Absorbance was measured with a microplate reader at a wavelength of 450 nm. The results are shown in Table 2.
TABLE 2
As is clear from the above table, the adipose-derived stem cell compositions for wound repair prepared in examples 1 to 3 of the present invention have low cytotoxicity.
Test example 3
Balb/c mice were randomly divided into 3 groups of 6, control (PBS solution), examples 1-3, and comparative examples 1-7, respectively. After the mice were anesthetized with 1% pentobarbital sodium, the backs were dehaired, washed with warm physiological saline, wiped dry, and iodine tincture was sterilized, and the whole skin with a diameter of about 1cm was cut off from the backs of the mice to prepare a wound model. 100. Mu.L of E.coli (1X 106 cfu/mL) in normal saline was inoculated onto the skin wound. After 1d infection, 200 mu L of adipose-derived stem cell composition for wound repair of the control group and each experimental group was placed on the back wound surface, the wound healing area and the healing time were observed and recorded on days 0 and 14 after the wound, the wound healing rate was calculated according to the following formula, and the wound healing speed was evaluated.
Wound healing rate = (day 0 wound area-14 th after day) day 0 wound area x 100%.
The results are shown in Table 3.
TABLE 3 Table 3
From the above table, the adipose-derived stem cell compositions for wound repair prepared in examples 1 to 3 of the present invention have a good function of healing skin wound surface.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.