Adipose stem cell composition for wound repair and preparation method and application thereof

Abstract

The present invention proposes a fat stem cell composition for wound repair and a preparation method and application thereof, which belongs to the field of stem cell technology. Fetal bovine serum, dexamethasone, penicillin, streptomycin, collagen, magnetic particles, and substance P are added to DMEM culture medium to prepare a culture medium for low-frequency magnetic field culture of fat mesenchymal stem cells, separation, obtaining a stem cell solution, embedding to obtain stem cell microspheres, freeze-drying the supernatant to obtain paracrine culture material, and mixing with stem cell microspheres, hydroxypropyl methylcellulose, carboxymethyl chitosan, hyaluronic acid, glycerol, and stem cell microspheres to obtain a fat stem cell composition for wound repair, which has good effects of implanting damaged tissues, releasing nutrients, promoting new blood vessel formation, regulating oxidative stress and inducing anti-inflammatory reactions, and is helpful for wound repair. It can be converted into a gel phase at a skin epidermal temperature between 35-37°C, stabilizes components, and releases in a controlled manner.

Description

Technical Field

[0001] The present invention relates to the field of stem cell technology, and in particular to an adipose stem cell composition for wound repair, and a preparation method and application thereof. Background Art

[0002] The skin is the largest organ in the human body. As a protective barrier, it plays an important role in protecting against external mechanical damage and preventing pathogenic microorganisms and chemical substances from invading the body. The mildest trauma is limited to the epidermis of the skin. Slightly more serious cases may cause rupture of the skin and subcutaneous tissue, and wounds. Severe trauma may cause rupture and fracture of muscles, tendons, nerves. Mild trauma can heal through epithelial regeneration. The latter two, due to the severity of the trauma, will cause changes in the appearance and histopathology of normal skin tissue and form scars. When scar growth exceeds a certain limit, various complications will occur, such as destruction of appearance and functional activity disorders, which will cause great physical and mental pain to patients, especially scars left after burns, scalds, and severe trauma.

[0003] Various traumas can cause varying degrees of cell degeneration, necrosis and tissue defects, and tissue repair must be carried out through cell proliferation and the formation of intercellular matrix. In this repair process, fibroblasts play a very important role. In the later stage of trauma repair, fibroblasts participate in the reconstruction of repaired tissues by secreting collagenase. Adipose-derived stem cells (ASCs) were first reported to be isolated from adipose tissue in 2001. Because they have the same high proliferation and self-renewal ability as bone marrow-derived stem cells (BMSCs), a large number of ASCs can be isolated from adipose tissue, and they can multidirectionally differentiate into mesoderm and non-mesoderm cells, making them a hot spot in the application of adult stem cells in regenerative medicine. They are widely used in many research fields such as skin tissue regeneration, cartilage and bone tissue regeneration, and wound healing.

[0004] In the research of wound repair, how to inhibit or even eliminate the formation of scars is a difficult problem in current clinical medicine. Chinese patent CN107320768A discloses an auxiliary material for wound repair containing induced pluripotent stem cell extract, which utilizes the ability of induced pluripotent stem cell extract to induce transformation of fibroblasts. Its main effect is to promote wound repair and inhibit bacteria, but its effect on severe skin wounds and burns is poor, especially in the process of repairing severe wounds, the inhibitory effect on scar production is poor.

[0005] Therefore, how to use adipose-derived mesenchymal stem cells (also called adipose-derived stem cells) to provide a topical composition for inhibiting or even eliminating scars has become an urgent problem to be solved in the prior art. Summary of the invention

[0006] The purpose of the present invention is to propose an adipose stem cell composition for wound repair, a preparation method and an application thereof, which has the effects of being well implanted into damaged tissues, releasing nutrients, promoting new blood vessel formation, regulating oxidative stress and inducing anti-inflammatory responses, and is helpful for wound repair. It can be transformed into a gel phase at a skin epidermal temperature between 35-37°C, stabilize components, and release in a slow and controlled manner.

[0007] The technical solution of the present invention is achieved in this way:

[0008] The invention provides a method for preparing an adipose stem cell composition for wound repair. The method comprises the following steps: adding fetal bovine serum, dexamethasone, penicillin, streptomycin, collagen, magnetic particles and substance P into a DMEM culture medium to prepare a culture medium, which is used for low-frequency magnetic field culture of adipose mesenchymal stem cells, separating the culture medium to obtain a stem cell solution, embedding the solution to obtain stem cell microspheres, freeze-drying the supernatant to obtain a paracrine culture substance, mixing the solution with stem cell microspheres, hydroxypropyl methylcellulose, carboxymethyl chitosan, hyaluronic acid, glycerol and the stem cell microspheres, stirring and uniformly mixing the solution to obtain the adipose stem cell composition for wound repair.

[0009] As a further improvement of the present invention, the following steps are included:

[0010] S1. Preparation of culture medium: adding fetal bovine serum, dexamethasone, penicillin, streptomycin, collagen, magnetic particles, and substance P to DMEM culture medium to prepare a culture medium;

[0011] S2. Culture of adipose-derived mesenchymal stem cells: After the adipose-derived mesenchymal stem cells are revived, they are added to the culture medium and cultured in a low-frequency magnetic field. After the cells adhere to the wall and grow to 80-90% fusion, the magnetic material is separated by a magnet, the residue is centrifuged, the supernatant is collected, the solid is washed with D-Hank's solution, digested, resuspended in DMEM culture medium, and frozen to obtain a stem cell solution;

[0012] S3 culture medium processing: the supernatant in step S2 is freeze-dried to obtain a paracrine culture material;

[0013] S4. Preparation of silk fibroin peptide scaffold particles: dissolving silk fibroin peptide and lecithin in water to obtain a solution, adding dropwise to liquid paraffin, emulsifying, adding a mixed solution of EDC and NHS, stirring and solidifying, centrifuging, washing, and drying to obtain silk fibroin peptide scaffold particles;

[0014] S5. Preparation of stem cell microspheres: After the stem cell solution is revived, silk fibroin peptide scaffold particles are added, stirred for adsorption, sodium alginate and lecithin are added, stirred and mixed evenly, added dropwise to liquid paraffin, emulsified, calcium chloride solution is added dropwise, solidified at room temperature, centrifuged, washed, and dried to obtain stem cell microspheres;

[0015] S6. Preparation of thermosensitive hydrogel: Dissolve hydroxypropyl methylcellulose in hot water, cool to room temperature, add carboxymethyl chitosan, hyaluronic acid and glycerol, stir and mix evenly, place at 4°C, add stem cell microspheres and paracrine culture material, stir and mix evenly, and prepare a fat stem cell composition for wound repair.

[0016] As a further improvement of the present invention, the content of fetal bovine serum in the culture medium in 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 100-120μg / mL, the content of collagen is 70-100μg / mL, the content of magnetic particles is 100-200μg / mL, the content of substance P is 3-5μg / mL, and the magnetic particles are micron-sized ferrosoferric oxide.

[0017] As a further improvement of the present invention, the low-frequency magnetic field culture time in step S2 is 1-2 weeks, the culture conditions are 36-38°C, the CO2 concentration is 3-6v / v%, the frequency of the low-frequency magnetic field is 40-50Hz, and the stimulation time is 30-40min / d; the digestive fluid is a PBS buffer solution with a pH of 7.5-7.7 containing 1-2wt% trypsin and 0.5-1wt% alkaline protease.

[0018] As a further improvement of the present invention, the mass ratio of the silk fibroin peptide, lecithin, EDC and NHS in step S4 is 15-20:0.5-1:0.1-0.2:0.15-0.25.

[0019] As a further improvement of the present invention, the mass ratio of the stem cell solution, the silk fibroin peptide scaffold particles, the sodium alginate and the lecithin in step S5 is 100:12-15:17-22:1-2.

[0020] As a further improvement of the present invention, the temperature of the hot water in step S6 is 70-80° C., and the mass ratio of the hydroxypropyl methylcellulose, carboxymethyl chitosan, hyaluronic acid, glycerol, stem cell microspheres and paracrine culture material is 10-12:5-7:3-4:2-4:7-10:4-6.

[0021] As a further improvement of the present invention, the present invention specifically comprises the following steps:

[0022] S1. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, collagen, micron-sized ferrosoferric oxide, and substance P were added to DMEM culture medium to prepare a culture medium;

[0023] The culture medium contains 9-11 wt % fetal bovine serum, 5-15 nmol / L dexamethasone, 100-120 U / mL penicillin, 100-120 μg / mL streptomycin, 70-100 μg / mL collagen, 100-200 μg / mL micron-grade ferrosoferric oxide, and 3-5 μg / mL substance P.

[0024] S2. Cultivation of adipose-derived mesenchymal stem cells: add adipose-derived mesenchymal stem cells to culture medium after recovery, culture for 1-2 weeks at 36-38°C, CO2 concentration of 3-6v / v%, and low-frequency magnetic field, wherein the frequency of the low-frequency magnetic field is 40-50Hz, and the stimulation time is 30-40min / d. After the cells adhere to the wall and grow to 80-90% fusion, separate the magnetic material with a magnet, centrifuge the residue, collect the supernatant, rinse the solid with D-Hank's solution, digest, resuspend in DMEM culture medium, and freeze to obtain a stem cell solution;

[0025] The digestion solution is a PBS buffer solution with a pH of 7.5-7.7 containing 1-2wt% trypsin and 0.5-1wt% alkaline protease;

[0026] S3 culture medium processing: the supernatant in step S2 is freeze-dried to obtain a paracrine culture material;

[0027] S4. Preparation of silk fibroin peptide scaffold particles: 15-20 parts by weight of silk fibroin peptide and 0.5-1 parts by weight of lecithin are dissolved in water to obtain a solution, which is added dropwise to liquid paraffin, emulsified, and 20 mL of a mixed solution containing 0.1-0.2 parts by weight of EDC and 0.15-0.25 parts by weight of NHS is added, stirred for solidification, centrifuged, washed, and dried to obtain silk fibroin peptide scaffold particles;

[0028] S5. Preparation of stem cell microspheres: After resuscitation of 100 parts by weight of stem cell solution, add 12-15 parts by weight of silk fibroin peptide scaffold particles, stir and adsorb, add 17-22 parts by weight of sodium alginate and 1-2 parts by weight of lecithin, stir and mix evenly, dropwise add to liquid paraffin, emulsify, dropwise add calcium chloride solution, solidify at room temperature, centrifuge, wash, and dry to obtain stem cell microspheres;

[0029] S6. Preparation of thermosensitive hydrogel: dissolve 10-12 parts by weight of hydroxypropyl methylcellulose in hot water at 70-80°C, cool to room temperature, add 5-7 parts by weight of carboxymethyl chitosan, 3-4 parts by weight of hyaluronic acid and 2-4 parts by weight of glycerol, stir and mix evenly, place at 4°C, add 7-10 parts by weight of stem cell microspheres and 4-6 parts by weight of paracrine culture material, stir and mix evenly, to obtain an adipose stem cell composition for wound repair.

[0030] The present invention further protects an adipose stem cell composition for wound repair prepared by the above preparation method.

[0031] The present invention further protects the use of the above-mentioned adipose stem cell composition for wound repair in the preparation of a medicine for repairing skin wounds.

[0032] The present invention has the following beneficial effects:

[0033] Wound healing is a highly complex biological process consisting of three overlapping stages: inflammation, proliferation, and remodeling. Damage at any one or more stages will lead to repair disorders. Adipose-derived mesenchymal stem cells can be implanted into damaged tissues, release nutrients, promote new blood vessel formation, regulate oxidative stress, and induce anti-inflammatory responses. Epidermal cells and fibroblasts play an important role in the repair of skin wounds, participating in multiple physiological processes such as activation, proliferation and re-epithelialization, wound contraction, tissue reconstruction, and extracellular matrix deposition of wound repair. Adipose-derived mesenchymal stem cells can directly differentiate into cell lines required for skin wound repair under the influence of the local microenvironment, such as epidermal cell lines, fibroblast cell lines, and endothelial cell lines. At the same time, they secrete growth factors to promote the proliferation and migration of epidermal cells, fibroblasts, and endothelial cells, thereby promoting skin wound healing.

[0034] Adding collagen to the culture medium is an important raw material for repairing tissue after skin damage, providing raw materials for skin repair. At the same time, collagen can also promote the proliferation and differentiation of stem cells, promote paracrine function, and increase the secretion of exosomes.

[0035] Micron-sized ferroferric oxide is a type of magnetic material. During the low-frequency magnetic field culture process, it adheres to the surface of stem cells to form a micro-magnetic field by responding to the magnetic field, affecting the structure of the cell membrane and cell membrane-related enzymes to increase the permeability of small molecules, thereby changing cell behavior with the help of the second messenger. At the same time, it controls the opening of voltage-gated calcium channels, causing calcium ion influx, increasing the intracellular calcium ion concentration, and promoting the growth and development of cells.

[0036] Substance P is a highly conserved neuropeptide, called "injury-inducing factor" by some researchers. It has the ability to regulate inflammation, recruit stem cells, promote cell proliferation and migration, and promote vascularization. At the same time, it can enhance the paracrine function of stem cells, and therefore, can play a synergistic role in promoting regeneration.

[0037] Silk protein peptide is a kind of protein peptide with good biocompatibility and good mechanical strength. Glutaraldehyde is a commonly used chemical crosslinking agent, but the crosslinking products of glutaraldehyde are usually released as toxic substances due to biodegradation, which is not conducive to skin wound repair. EDC and NHS can help form amide bonds between carboxyl and amino groups in silk protein peptide molecules, but they themselves do not become part of the actual crosslinking. They are non-toxic and have good biocompatibility, thereby improving the safety and biocompatibility of the prepared products. Through the hydrogen bond adsorption between protein peptides and glycoproteins on the surface of stem cells, stem cells can be adsorbed and fixed on silk protein peptide scaffold particles, and then coated in sodium alginate microspheres, replacing water molecules to form hydration bonds with hydrophilic groups on cell membranes and proteins, avoiding changes in the structure of cell membranes and protein molecules, maintaining the stability of their structure and function, allowing stem cells to be stored for a long time, improving their storage stability, and also improving the storage stability of exosomes.

[0038] Hydroxypropyl methylcellulose is a thermosensitive hydrophilic polymer derived from cellulose. It has excellent properties such as non-toxicity, low cost, high swelling and surface activity. The hydrogel prepared by mixing it with glycerol and hyaluronic acid has thermosensitive properties and can undergo sol-gel phase transition according to changes in ambient temperature. Paracrine culture material contains collagen, which can form thermosensitive hydrogels with chitosan. The introduction of collagen can shorten the gel time and improve the cross-linking degree, mechanical strength and biocompatibility of the hydrogel. By mixing two different hydrogel systems, the phase transition temperature of the hydrogel can be adjusted under the interaction, so that it can be converted into a gel phase between 35-37°C of the skin epidermis temperature, and a solution phase below this temperature, which makes it easy to coat and form a gel on the wound surface, slowly release drugs, isolate oxygen and promote rapid healing and recovery of the wound.

[0039] The adipose stem cell composition for wound repair prepared by the present invention has the effects of better implantation into damaged tissues, releasing nutrients, promoting new blood vessel formation, regulating oxidative stress and inducing anti-inflammatory response, which is helpful for wound repair. It can be transformed into a gel phase at a skin epidermal temperature between 35-37°C, stabilize components, and release slowly and in a controlled manner. DETAILED DESCRIPTION

[0040] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] Collagen, collagen tripeptide, 280-300Da, content>99%, purchased from Yancheng Runyu Biotechnology Co., Ltd. Micron-grade ferroferric oxide, 200 mesh, purchased from Qinghe Nuotu Welding Materials Co., Ltd. P substance, CAS number: 33507-63-0, content>98%, brand Widely, purchased from Hubei Weideli Chemical Reagent Co., Ltd. Trypsin, 200,000 U / g, alkaline protease, 200,000 U / g, purchased from Xiasheng (Beijing) Enzyme Biotechnology Development Co., Ltd.

[0042] NHS, N-hydroxysuccinimide; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide. Example 1

[0043] This embodiment provides a method for preparing an adipose stem cell composition for wound repair, which specifically comprises the following steps:

[0044] S1. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, collagen, micron-sized ferrosoferric oxide, and substance P were added to DMEM culture medium to prepare a culture medium;

[0045] The culture medium contains 9 wt % fetal bovine serum, 5 nmol / L dexamethasone, 100 U / mL penicillin, 100 μg / mL streptomycin, 70 μg / mL collagen, 100 μg / mL micron-sized ferrosoferric oxide, and 3 μg / mL substance P.

[0046] S2. Culture of adipose-derived mesenchymal stem cells: add adipose-derived mesenchymal stem cells to culture medium after recovery, and culture them for 1 week at 36°C with a CO2 concentration of 3v / v%. The frequency of the low-frequency magnetic field is 40 Hz, and the stimulation time is 30 min / d. After the cells adhere to the wall and grow to 80-90% fusion, separate the magnetic material with a magnet, centrifuge the residue, collect the supernatant, rinse the solid with D-Hank's solution, digest with digestion solution, resuspend in DMEM culture medium, and freeze to obtain a stem cell solution.

[0047] The digestion solution is a PBS buffer with a pH of 7.5 containing 1wt% trypsin and 0.5wt% alkaline protease;

[0048] S3 culture medium processing: the supernatant in step S2 is freeze-dried to obtain a paracrine culture material;

[0049] S4. Preparation of silk fibroin peptide scaffold particles: 15 g of silk fibroin peptide and 0.5 g of lecithin were dissolved in 300 mL of water to obtain a solution, which was added dropwise to 500 mL of liquid paraffin, emulsified at 7000 r / min for 15 min, and 20 mL of a mixed solution containing 0.1 g of EDC and 0.15 g of NHS was added, stirred and solidified for 2 h, centrifuged, washed, and dried to obtain silk fibroin peptide scaffold particles;

[0050] S5. Preparation of stem cell microspheres: After resuscitation of 100 g of stem cell solution, add 12 g of silk fibroin peptide scaffold particles, stir and adsorb for 20 min, add 17 g of sodium alginate and 1 g of lecithin, stir and mix for 10 min, add dropwise to 300 mL of liquid paraffin, emulsify at 7000 r / min for 15 min, add dropwise 20 mL of 5 wt% calcium chloride solution, solidify at room temperature for 30 min, centrifuge, wash, and dry to obtain stem cell microspheres;

[0051] S6. Preparation of thermosensitive hydrogel: Dissolve 10 g of hydroxypropyl methylcellulose in 500 mL of 70°C hot water, cool to room temperature, add 5 g of carboxymethyl chitosan, 3 g of hyaluronic acid and 2 g of glycerol, stir and mix for 15 minutes, place at 4°C, add 7 g of stem cell microspheres and 4 g of paracrine culture material, stir and mix for 15 minutes to obtain an adipose stem cell composition for wound repair. Example 2

[0052] This embodiment provides a method for preparing an adipose stem cell composition for wound repair, which specifically comprises the following steps:

[0053] S1. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, collagen, micron-sized ferrosoferric oxide, and substance P were added to DMEM culture medium to prepare a culture medium;

[0054] The culture medium contains 11 wt % fetal bovine serum, 15 nmol / L dexamethasone, 120 U / mL penicillin, 120 μg / mL streptomycin, 100 μg / mL collagen, 200 μg / mL micron-grade ferrosoferric oxide, and 5 μg / mL substance P.

[0055] S2. Culture of adipose-derived mesenchymal stem cells: add adipose-derived mesenchymal stem cells to culture medium after recovery, and culture them in a low-frequency magnetic field at 38°C and a CO2 concentration of 6v / v% for 2 weeks. The frequency of the low-frequency magnetic field is 50 Hz, and the stimulation time is 40 min / d. After the cells adhere to the wall and grow to 80-90% fusion, separate the magnetic material with a magnet, centrifuge the residue, collect the supernatant, rinse the solid with D-Hank's solution, digest with digestion solution, resuspend in DMEM culture medium, and freeze to obtain a stem cell solution;

[0056] The digestion solution is a PBS buffer solution with a pH of 7.7 containing 2wt% trypsin and 1wt% alkaline protease;

[0057] S3 culture medium processing: the supernatant in step S2 is freeze-dried to obtain a paracrine culture material;

[0058] S4. Preparation of silk fibroin peptide scaffold particles: 20 g of silk fibroin peptide and 1 g of lecithin were dissolved in 300 mL of water to obtain a solution, which was added dropwise to 500 mL of liquid paraffin, emulsified at 7000 r / min for 15 min, and 20 mL of a mixed solution containing 0.2 g of EDC and 0.25 g of NHS was added, stirred and solidified for 2 h, centrifuged, washed, and dried to obtain silk fibroin peptide scaffold particles;

[0059] S5. Preparation of stem cell microspheres: After resuscitation of 100 g of stem cell solution, add 15 g of silk fibroin peptide scaffold particles, stir and adsorb for 20 min, add 22 g of sodium alginate and 2 g of lecithin, stir and mix for 10 min, add dropwise to 300 mL of liquid paraffin, emulsify at 7000 r / min for 15 min, add dropwise 20 mL of 5 wt% calcium chloride solution, solidify at room temperature for 30 min, centrifuge, wash, and dry to obtain stem cell microspheres;

[0060] S6. Preparation of thermosensitive hydrogel: Dissolve 12 g of hydroxypropyl methylcellulose in 500 mL of 80°C hot water, cool to room temperature, add 7 g of carboxymethyl chitosan, 4 g of hyaluronic acid and 4 g of glycerol, stir and mix for 15 minutes, place at 4°C, add 10 g of stem cell microspheres and 6 g of paracrine culture material, stir and mix for 15 minutes to obtain an adipose stem cell composition for wound repair. Example 3

[0061] This embodiment provides a method for preparing an adipose stem cell composition for wound repair, which specifically comprises the following steps:

[0062] S1. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, collagen, micron-sized ferrosoferric oxide, and substance P were added to DMEM culture medium to prepare a culture medium;

[0063] The culture medium contains 10 wt % fetal bovine serum, 10 nmol / L dexamethasone, 110 U / mL penicillin, 110 μg / mL streptomycin, 85 μg / mL collagen, 150 μg / mL micron-grade ferrosoferric oxide, and 4 μg / mL substance P.

[0064] S2. Culture of adipose-derived mesenchymal stem cells: add adipose-derived mesenchymal stem cells to culture medium after recovery, and culture them in a low-frequency magnetic field at 37°C and a CO2 concentration of 5v / v% for 2 weeks. The frequency of the low-frequency magnetic field is 45Hz, and the stimulation time is 35min / d. After the cells adhere to the wall and grow to 80-90% fusion, separate the magnetic material with a magnet, centrifuge the residue, collect the supernatant, rinse the solid with D-Hank's solution, digest with digestion solution, resuspend in DMEM culture medium, and freeze to obtain a stem cell solution;

[0065] The digestion solution is a PBS buffer with a pH of 7.6 containing 1.5wt% trypsin and 0.7wt% alkaline protease;

[0066] S3 culture medium processing: the supernatant in step S2 is freeze-dried to obtain a paracrine culture material;

[0067] S4. Preparation of silk fibroin peptide scaffold particles: 17 g of silk fibroin peptide and 0.7 g of lecithin were dissolved in 300 mL of water to obtain a solution, which was added dropwise to 500 mL of liquid paraffin, emulsified at 7000 r / min for 15 min, and 20 mL of a mixed solution containing 0.15 g of EDC and 0.2 g of NHS was added, stirred and solidified for 2 h, centrifuged, washed, and dried to obtain silk fibroin peptide scaffold particles;

[0068] S5. Preparation of stem cell microspheres: After resuscitation of 100 g of stem cell solution, add 13 g of silk fibroin peptide scaffold particles, stir and adsorb for 20 min, add 20 g of sodium alginate and 1.5 g of lecithin, stir and mix for 10 min, dropwise add to 300 mL of liquid paraffin, emulsify at 7000 r / min for 15 min, dropwise add 20 mL of 5 wt% calcium chloride solution, solidify at room temperature for 30 min, centrifuge, wash, and dry to obtain stem cell microspheres;

[0069] S6. Preparation of thermosensitive hydrogel: Dissolve 11 g of hydroxypropyl methylcellulose in 500 mL of 75°C hot water, cool to room temperature, add 6 g of carboxymethyl chitosan, 3.5 g of hyaluronic acid and 3 g of glycerol, stir and mix for 15 minutes, place at 4°C, add 8 g of stem cell microspheres and 5 g of paracrine culture material, stir and mix for 15 minutes to obtain an adipose stem cell composition for wound repair.

[0070] Comparative Example 1

[0071] Compared with Example 3, the difference is that collagen is not added in step S1.

[0072] The details are as follows:

[0073] S1. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, micron-sized ferrosoferric oxide, and substance P were added to DMEM culture medium to prepare a culture medium;

[0074] The culture medium contains 10 wt % fetal bovine serum, 10 nmol / L dexamethasone, 110 U / mL penicillin, 110 μg / mL streptomycin, 150 μg / mL micron-grade ferrosoferric oxide, and 4 μg / mL substance P.

[0075] Comparative Example 2

[0076] Compared with Example 3, the difference is that micron-sized ferrosoferric oxide is not added in step S1.

[0077] The details are as follows:

[0078] S1. Preparation of culture medium: adding fetal bovine serum, dexamethasone, penicillin, streptomycin, collagen, and substance P to DMEM culture medium to prepare a culture medium;

[0079] The culture medium contains 10 wt % fetal bovine serum, 10 nmol / L dexamethasone, 110 U / mL penicillin, 110 μg / mL streptomycin, 85 μg / mL collagen and 4 μg / mL substance P.

[0080] Comparative Example 3

[0081] Compared with Example 3, the difference is that no P substance is added in step S1.

[0082] The details are as follows:

[0083] S1. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, collagen, and micron-sized ferrosoferric oxide were added to DMEM culture medium to prepare a culture medium;

[0084] The culture medium contains 10 wt % fetal bovine serum, 10 nmol / L dexamethasone, 110 U / mL penicillin, 110 μg / mL streptomycin, 85 μg / mL collagen, and 150 μg / mL micron-grade ferrosoferric oxide.

[0085] Comparative Example 4

[0086] Compared with Example 3, the difference is that low-frequency magnetic field cultivation is not performed in step S2.

[0087] The details are as follows:

[0088] S2. Culture of adipose-derived mesenchymal stem cells: add adipose-derived mesenchymal stem cells to culture medium after recovery, and culture at 37°C with a CO2 concentration of 5v / v% for 2 weeks. After the cells adhere to the wall and grow to 80-90% fusion, separate the magnetic material with a magnet, centrifuge the residue, collect the supernatant, wash the solid with D-Hank's solution, digest with digestion solution, resuspend in DMEM culture medium, and freeze to prepare a stem cell solution;

[0089] The digestion solution is a PBS buffer solution with a pH of 7.6 and containing 1.5 wt % trypsin and 0.7 wt % alkaline protease.

[0090] Comparative Example 5

[0091] Compared with Example 3, the difference is that step S4 is not performed.

[0092] The details are as follows:

[0093] S1. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, collagen, micron-sized ferrosoferric oxide, and substance P were added to DMEM culture medium to prepare a culture medium;

[0094] The culture medium contains 10 wt % fetal bovine serum, 10 nmol / L dexamethasone, 110 U / mL penicillin, 110 μg / mL streptomycin, 85 μg / mL collagen, 150 μg / mL micron-grade ferrosoferric oxide, and 4 μg / mL substance P.

[0095] S2. Culture of adipose-derived mesenchymal stem cells: add adipose-derived mesenchymal stem cells to culture medium after recovery, and culture them in a low-frequency magnetic field at 37°C and a CO2 concentration of 5v / v% for 2 weeks. The frequency of the low-frequency magnetic field is 45Hz, and the stimulation time is 35min / d. After the cells adhere to the wall and grow to 80-90% fusion, separate the magnetic material with a magnet, centrifuge the residue, collect the supernatant, rinse the solid with D-Hank's solution, digest with digestion solution, resuspend in DMEM culture medium, and freeze to obtain a stem cell solution;

[0096] The digestion solution is a PBS buffer with a pH of 7.6 containing 1.5wt% trypsin and 0.7wt% alkaline protease;

[0097] S3 culture medium processing: the supernatant in step S2 is freeze-dried to obtain a paracrine culture material;

[0098] S4. Preparation of stem cell microspheres: After resuscitation of 100 g of stem cell solution, add 20 g of sodium alginate and 1.5 g of lecithin, stir and mix for 10 min, add dropwise to 300 mL of liquid paraffin, emulsify at 7000 r / min for 15 min, add dropwise 20 mL of 5 wt% calcium chloride solution, solidify at room temperature for 30 min, centrifuge, wash, and dry to obtain stem cell microspheres;

[0099] S5. Preparation of thermosensitive hydrogel: Dissolve 11 g of hydroxypropyl methylcellulose in 500 mL of 75°C hot water, cool to room temperature, add 6 g of carboxymethyl chitosan, 3.5 g of hyaluronic acid and 3 g of glycerol, stir and mix for 15 minutes, place at 4°C, add 8 g of stem cell microspheres and 5 g of paracrine culture material, stir and mix for 15 minutes to obtain an adipose stem cell composition for wound repair.

[0100] Comparative Example 6

[0101] Compared with Example 3, the difference is that step S5 embedding is not performed.

[0102] The details are as follows:

[0103] S1. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, collagen, micron-sized ferrosoferric oxide, and substance P were added to DMEM culture medium to prepare a culture medium;

[0104] The culture medium contains 10 wt % fetal bovine serum, 10 nmol / L dexamethasone, 110 U / mL penicillin, 110 μg / mL streptomycin, 85 μg / mL collagen, 150 μg / mL micron-grade ferrosoferric oxide, and 4 μg / mL substance P.

[0105] S2. Culture of adipose-derived mesenchymal stem cells: add adipose-derived mesenchymal stem cells to culture medium after recovery, and culture them in a low-frequency magnetic field at 37°C and a CO2 concentration of 5v / v% for 2 weeks. The frequency of the low-frequency magnetic field is 45Hz, and the stimulation time is 35min / d. After the cells adhere to the wall and grow to 80-90% fusion, separate the magnetic material with a magnet, centrifuge the residue, collect the supernatant, rinse the solid with D-Hank's solution, digest with digestion solution, resuspend in DMEM culture medium, and freeze to obtain a stem cell solution;

[0106] The digestion solution is a PBS buffer with a pH of 7.6 containing 1.5wt% trypsin and 0.7wt% alkaline protease;

[0107] S3 culture medium processing: the supernatant in step S2 is freeze-dried to obtain a paracrine culture material;

[0108] S4. Preparation of silk fibroin peptide scaffold particles: 17 g of silk fibroin peptide and 0.7 g of lecithin were dissolved in 300 mL of water to obtain a solution, which was added dropwise to 500 mL of liquid paraffin, emulsified at 7000 r / min for 15 min, and 20 mL of a mixed solution containing 0.15 g of EDC and 0.2 g of NHS was added, stirred and solidified for 2 h, centrifuged, washed, and dried to obtain silk fibroin peptide scaffold particles;

[0109] S5. Preparation of stem cell particles: After resuscitation of 100 g of stem cell solution, 13 g of silk fibroin peptide scaffold particles were added, and the mixture was stirred and adsorbed for 20 min to obtain stem cell particles;

[0110] S6. Preparation of thermosensitive hydrogel: Dissolve 11 g of hydroxypropyl methylcellulose in 500 mL of 75°C hot water, cool to room temperature, add 6 g of carboxymethyl chitosan, 3.5 g of hyaluronic acid and 3 g of glycerol, stir and mix for 15 minutes, place at 4°C, add 8 g of stem cell particles and 5 g of paracrine culture material, stir and mix for 15 minutes to obtain an adipose stem cell composition for wound repair.

[0111] Comparative Example 7

[0112] Compared with the embodiment 3, the difference is that step S6 is not performed.

[0113] The details are as follows:

[0114] S1. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, collagen, micron-sized ferrosoferric oxide, and substance P were added to DMEM culture medium to prepare a culture medium;

[0115] The culture medium contains 10 wt % fetal bovine serum, 10 nmol / L dexamethasone, 110 U / mL penicillin, 110 μg / mL streptomycin, 85 μg / mL collagen, 150 μg / mL micron-grade ferrosoferric oxide, and 4 μg / mL substance P.

[0116] S2. Culture of adipose-derived mesenchymal stem cells: add adipose-derived mesenchymal stem cells to culture medium after recovery, and culture them in a low-frequency magnetic field at 37°C and a CO2 concentration of 5v / v% for 2 weeks. The frequency of the low-frequency magnetic field is 45Hz, and the stimulation time is 35min / d. After the cells adhere to the wall and grow to 80-90% fusion, separate the magnetic material with a magnet, centrifuge the residue, collect the supernatant, rinse the solid with D-Hank's solution, digest with digestion solution, resuspend in DMEM culture medium, and freeze to obtain a stem cell solution;

[0117] The digestion solution is a PBS buffer with a pH of 7.6 containing 1.5wt% trypsin and 0.7wt% alkaline protease;

[0118] S3 culture medium processing: the supernatant in step S2 is freeze-dried to obtain a paracrine culture material;

[0119] S4. Preparation of silk fibroin peptide scaffold particles: 17 g of silk fibroin peptide and 0.7 g of lecithin were dissolved in 300 mL of water to obtain a solution, which was added dropwise to 500 mL of liquid paraffin, emulsified at 7000 r / min for 15 min, and 20 mL of a mixed solution containing 0.15 g of EDC and 0.2 g of NHS was added, stirred and solidified for 2 h, centrifuged, washed, and dried to obtain silk fibroin peptide scaffold particles;

[0120] S5. Preparation of stem cell microspheres: After resuscitation of 100 g of stem cell solution, add 13 g of silk fibroin peptide scaffold particles, stir and adsorb for 20 min, add 20 g of sodium alginate and 1.5 g of lecithin, stir and mix for 10 min, dropwise add to 300 mL of liquid paraffin, emulsify at 7000 r / min for 15 min, dropwise add 20 mL of 5 wt% calcium chloride solution, solidify at room temperature for 30 min, centrifuge, wash, and dry to obtain stem cell microspheres;

[0121] S6. Preparation of an adipose-derived stem cell composition for wound repair: 8 g of stem cell microspheres and 5 g of paracrine culture material were stirred and mixed for 15 min to prepare an adipose-derived stem cell composition for wound repair.

[0122] Comparative Example 8

[0123] Compared with the embodiment 3, the difference is that step S6 is different.

[0124] The details are as follows:

[0125] S6. Preparation of thermosensitive hydrogel: Add 6 g of carboxymethyl chitosan and 3.5 g of hyaluronic acid into 500 mL of water, stir and mix for 15 min, place at 4°C, add 8 g of stem cell particles and 5 g of paracrine culture material, stir and mix for 15 min, and prepare an adipose stem cell composition for wound repair.

[0126] Comparative Example 9

[0127] Compared with the embodiment 3, the difference is that step S6 is different.

[0128] The details are as follows:

[0129] S6. Preparation of thermosensitive hydrogel: Dissolve 11 g of hydroxypropyl methylcellulose in 500 mL of 75°C hot water, cool to room temperature, add 3.5 g of hyaluronic acid and 3 g of glycerol, stir and mix for 15 minutes, place at 4°C, add 8 g of stem cell particles and 5 g of paracrine culture material, stir and mix for 15 minutes to obtain an adipose stem cell composition for wound repair.

[0130] Comparative Example 10

[0131] Compared with Example 3, the difference is that glutaraldehyde is used instead of EDC and NHS in step S4.

[0132] The details are as follows:

[0133] S4. Preparation of silk fibroin peptide scaffold particles: 17 g of silk fibroin peptide and 0.7 g of lecithin were dissolved in 300 mL of water to obtain a solution, which was added dropwise into 500 mL of liquid paraffin, emulsified at 7000 r / min for 15 min, and 20 mL of an aqueous solution containing 0.35 g of glutaraldehyde was added. The mixture was stirred and solidified for 2 h, centrifuged, washed, and dried to obtain silk fibroin peptide scaffold particles.

[0134] Test Example 1

[0135] The adipose stem cell compositions for wound repair prepared in Examples 1-3 and Comparative Examples 8 and 9 were placed in glass test tubes and placed in a constant temperature water bath. The test tubes were taken out every 10 minutes and inverted to observe whether the hydrogel flowed. If it flowed, the temperature was continued to rise at a rate of 1°C. If it did not flow, this temperature was defined as the gelation temperature T of the hydrogel. gel The results are shown in Table 1.

[0136] Table 1

[0137]

[0138] It can be seen from the above table that the adipose stem cell compositions for wound repair prepared in Examples 1-3 of the present invention have a relatively suitable gelation temperature.

[0139] Test Example 2

[0140] Take equal volumes of the adipose stem cell compositions for wound repair prepared in Examples 1-3 and Comparative Example 10 and add them into 10 mL of DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin as sample culture medium. 4The cells were inoculated into 96-well plates at a density of 100 μL / mL and cultured in a 37°C incubator containing 5% CO2 for 12 hours until the cells were completely attached. The culture medium of each group was discarded, and the experimental group was added with sample culture medium, and the control group was added with DMEM complete culture medium. Six parallel wells were set up in each group. On the 5th day, the culture plate was removed, 10 μL CCK-solution (CCK-8 kit, Dojindo, Japan) was added to each well, and incubated at 37°C in the dark for 2 hours. The absorbance was measured at a wavelength of 450 nm using an enzyme reader. The results are shown in Table 2.

[0141] Table 2

[0142]

[0143] It can be seen from the above table that the adipose stem cell compositions for wound repair prepared in Examples 1-3 of the present invention have low cytotoxicity.

[0144] Test Example 3

[0145] Balb / c mice were randomly divided into 3 groups, each with 6 mice, namely control group (PBS solution), Example 1-3 group, and Comparative Example 1-7 group. The mice were anesthetized with 1% sodium pentobarbital intraperitoneally, and the backs were depilated, rinsed with warm saline, wiped dry, and disinfected with iodine tincture. The full-thickness skin with a diameter of about 1 cm was cut off from the back of the mice to prepare a trauma model. 100 μL of PBS dissolved in saline was added. E.coli (1×10 6 cfu / mL) were inoculated on the skin wound. One day after infection, 200 μL of the adipose stem cell composition for wound repair in the control group and each experimental group was placed on the back wound. The wound healing area and healing time were observed and recorded on the 0th and 14th days after the wound, and the wound healing rate was calculated according to the following formula to evaluate the wound healing speed.

[0146] Wound healing rate = (wound area on day 0 - area after day 14) / wound area on day 0 × 100%.

[0147] The results are shown in Table 3.

[0148] Table 3

[0149]

[0150] It can be seen from the above table that the adipose stem cell compositions for wound repair prepared in Examples 1-3 of the present invention have a good function of healing skin wounds.

[0151] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an adipose stem cell composition for wound repair, characterized in that: The following steps are involved: S1. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, collagen, magnetic particles, and substance P are added to DMEM culture medium to prepare a 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 100-120μg / mL, the content of collagen is 70-100μg / mL, the content of magnetic particles is 100-200μg / mL, the content of substance P is 3-5μg / mL, the magnetic particles are micron-sized ferrosoferric oxide; the collagen is collagen tripeptide; S2. Culture of adipose-derived mesenchymal stem cells: After the adipose-derived mesenchymal stem cells are revived, they are added to the culture medium and cultured in a low-frequency magnetic field. After the cells adhere to the wall and grow to 80-90% fusion, the magnetic material is separated by a magnet, the residue is centrifuged, the supernatant is collected, the solid is washed with D-Hank's solution, digested, resuspended in DMEM culture medium, and frozen to obtain a stem cell solution; S3 culture medium processing: the supernatant in step S2 is freeze-dried to obtain a paracrine culture material; S4. Preparation of silk fibroin peptide scaffold particles: dissolving silk fibroin peptide and lecithin in water to obtain a solution, adding dropwise to liquid paraffin, emulsifying, adding a mixed solution of EDC and NHS, stirring and solidifying, centrifuging, washing, and drying to obtain silk fibroin peptide scaffold particles; S5. Preparation of stem cell microspheres: After the stem cell solution is revived, silk fibroin peptide scaffold particles are added, stirred for adsorption, sodium alginate and lecithin are added, stirred and mixed evenly, added dropwise to liquid paraffin, emulsified, calcium chloride solution is added dropwise, solidified at room temperature, centrifuged, washed, and dried to obtain stem cell microspheres; S6. Preparation of thermosensitive hydrogel: dissolve hydroxypropyl methylcellulose in hot water, cool to room temperature, add carboxymethyl chitosan, hyaluronic acid and glycerol, stir and mix evenly, place at 4°C, add stem cell microspheres and paracrine culture material, stir and mix evenly to obtain an adipose stem cell composition for wound repair, wherein the temperature of the hot water is 70-80°C.

2. The preparation method according to claim 1, characterized in that: The low-frequency magnetic field culture time in step S2 is 1-2 weeks, the culture conditions are 36-38°C, the CO2 concentration is 3-6v / v%, the frequency of the low-frequency magnetic field is 40-50Hz, and the stimulation time is 30-40min / d; the digestion solution is a PBS buffer solution with a pH of 7.5-7.7 containing 1-2wt% trypsin and 0.5-1wt% alkaline protease.

3. The preparation method according to claim 1, characterized in that: The mass ratio of the silk fibroin peptide, lecithin, EDC and NHS in step S4 is 15-20:0.5-1:0.1-0.2:0.15-0.

25.

4. The preparation method according to claim 1, characterized in that: The mass ratio of the stem cell solution, the silk fibroin peptide scaffold particles, the sodium alginate and the lecithin in step S5 is 100:12-15:17-22:1-2.

5. The preparation method according to claim 1, characterized in that: The mass ratio of hydroxypropyl methylcellulose, carboxymethyl chitosan, hyaluronic acid, glycerol, stem cell microspheres and paracrine culture material in step S6 is 10-12:5-7:3-4:2-4:7-10:4-6.

6. The preparation method according to claim 1, characterized in that: The specific steps include: S1. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, collagen, micron-sized ferrosoferric oxide, and substance P were added to DMEM culture medium to prepare a culture medium; The culture medium contains 9-11 wt % fetal bovine serum, 5-15 nmol / L dexamethasone, 100-120 U / mL penicillin, 100-120 μg / mL streptomycin, 70-100 μg / mL collagen, 100-200 μg / mL micron-grade ferrosoferric oxide, and 3-5 μg / mL substance P. S2. Cultivation of adipose-derived mesenchymal stem cells: add adipose-derived mesenchymal stem cells to culture medium after recovery, culture for 1-2 weeks at 36-38°C, CO2 concentration of 3-6v / v%, and low-frequency magnetic field, wherein the frequency of the low-frequency magnetic field is 40-50Hz, and the stimulation time is 30-40min / d. After the cells adhere to the wall and grow to 80-90% fusion, separate the magnetic material with a magnet, centrifuge the residue, collect the supernatant, rinse the solid with D-Hank's solution, digest, resuspend in DMEM culture medium, and freeze to obtain a stem cell solution; The digestion solution is a PBS buffer with a pH of 7.5-7.7 containing 1-2wt% trypsin and 0.5-1wt% alkaline protease; S3 culture medium processing: the supernatant in step S2 is freeze-dried to obtain a paracrine culture material; S4. Preparation of silk fibroin peptide scaffold particles: 15-20 parts by weight of silk fibroin peptide and 0.5-1 parts by weight of lecithin are dissolved in water to obtain a solution, which is added dropwise to liquid paraffin, emulsified, and 20 mL of a mixed solution containing 1-2 parts by weight of EDC and 1.5-2.5 parts by weight of NHS is added, stirred for solidification, centrifuged, washed, and dried to obtain silk fibroin peptide scaffold particles; S5. Preparation of stem cell microspheres: After resuscitation of 100 parts by weight of stem cell solution, add 12-15 parts by weight of silk fibroin peptide scaffold particles, stir and adsorb, add 17-22 parts by weight of sodium alginate and 1-2 parts by weight of lecithin, stir and mix evenly, dropwise add to liquid paraffin, emulsify, dropwise add calcium chloride solution, solidify at room temperature, centrifuge, wash, and dry to obtain stem cell microspheres; S6. Preparation of thermosensitive hydrogel: dissolve 10-12 parts by weight of hydroxypropyl methylcellulose in hot water at 70-80°C, cool to room temperature, add 5-7 parts by weight of carboxymethyl chitosan, 3-4 parts by weight of hyaluronic acid and 2-4 parts by weight of glycerol, stir and mix evenly, place at 4°C, add 7-10 parts by weight of stem cell microspheres and 4-6 parts by weight of paracrine culture material, stir and mix evenly, to obtain an adipose stem cell composition for wound repair.

7. An adipose stem cell composition for wound repair obtained by the preparation method according to any one of claims 1 to 6.

8. Use of the adipose stem cell composition for wound repair as claimed in claim 7 in the preparation of a medicine for repairing skin wounds.

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