Hydrogel of supernatant of mesenchymal stem cells cultured without serum in load and its preparation method and application

Abstract

The present invention belongs to the technical field of biogels, and specifically relates to a hydrogel loaded with serum-free cultured mesenchymal stem cell supernatant, its preparation method and application. The hydrogel is prepared by mixing sodium hyaluronate, mesenchymal stem cell supernatant and poloxamer-127. The mass concentration of sodium hyaluronate in every 100 mL of the mesenchymal stem cell supernatant is 0.3 g to 2 g, and the mass concentration of poloxamer-127 is 20 g to 30 g. When culturing adipose mesenchymal stem cells, the present invention uses a serum-free medium for culture, and loads the cytokines in the supernatant onto the gel to extend the residence time of the supernatant.

Description

Technical Field

[0001] The present invention relates to the technical field of biogels, and particularly to a hydrogel loaded with serum-free mesenchymal stem cell supernatant, a preparation method thereof, and an application thereof. Background Art

[0002] Wound healing is a significantly dynamic and complex process that requires activation and coordination of cellular pathways to regulate inflammation, proliferation, and remodeling in order to restore tissue homeostasis and integrity after injury. Given the complexity of this physiological process, there has been a great deal of attention paid to the pathological problems caused by impaired wound healing, such as ischemia, bacterial colonization, senescence, and fibrotic regeneration. If the skin integrity is damaged, a wound dressing plays a role in promoting timely and healthy wound healing. A modern ideal wound dressing should be able to prevent wound infection, absorb excess exudate, and create a moist wound healing environment. Hydrogels are similar to natural extracellular matrix (ECM), capable of retaining a moist wound environment and enhancing autolytic debridement. With the development of modern medical science and technology, many new wound healing materials have been created. Chitosan, collagen, alginate, polypeptides, and polyurethane are commonly used biomaterials for wound healing applications. However, these commonly used materials have various limitations, which is a problem for chemically sensitive protein therapeutics that are prone to denaturation and degradation. Since the effectiveness and function of these regenerative therapies are entirely based on maintaining the structural-activity relationship of proteins, it is crucial to protect them from structural damage during encapsulation and delivery.

[0003] Mesenchymal stem cells are derived from the mesoderm and can differentiate into various cell types such as osteoblasts, chondrocytes, and adipocytes. After in vitro expansion, they can treat various diseases such as autoimmune diseases like psoriasis, osteoarthritis, anal fistula, amyotrophic lateral sclerosis, etc. A large number of cytokines are contained in their cell supernatant, including proteins such as COL1A1, COL1A2, FN1, TNC, COL4A, etc., which are proteins that play important functions during the wound recovery process. The cells are continuously cultured in vitro and then subjected to decellularization treatment, leaving the extracellular matrix (ECM) secreted by the cells. The extracellular matrix secreted by the cells contains sodium hyaluronate and collagen, which can provide a good microenvironment to promote the proliferation of fibroblasts and form an endogenous scaffold using the rich extracellular matrix secreted by the cells, thus avoiding various drawbacks brought by exogenous scaffolds.

[0004] Generally, in the method of culturing animal mesenchymal stem cells, serum needs to be added. Without serum, most cells cannot proliferate. Currently, the commonly used fetal bovine serum for serum has been found to have a risk of contaminating exogenous viruses and pathogenic factors, being prone to infecting the wound surface, and unable to solve the problem of the short residence time of mesenchymal stem cell cytokines at the wound site. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a hydrogel loaded with mesenchymal stem cell supernatant, a preparation method thereof, and an application thereof.

[0006] A preparation method of a hydrogel loaded with mesenchymal stem cell supernatant, comprising the following steps:

[0007] Culturing mesenchymal stem cells with a serum-free medium to obtain mesenchymal stem cell supernatant;

[0008] Mixing the mesenchymal stem cell supernatant with sodium hyaluronate and poloxamer-127 to obtain the hydrogel; the mass concentration of sodium hyaluronate in every 100 mL of the mesenchymal stem cell supernatant is 0.3 g to 2.8 g; the mass concentration of poloxamer-127 in every 100 mL of the mesenchymal stem cell supernatant is 20 g to 30 g. When culturing adipose mesenchymal stem cells in the present invention, a serum-free medium is used for culturing to avoid infection, and the cytokines in the supernatant are loaded onto the gel to prolong the residence time of the supernatant in vivo.

[0009] Preferably, the mass concentration of sodium hyaluronate in every 100 mL of the mesenchymal stem cell supernatant is 0.3 g.

[0010] Preferably, the mesenchymal stem cells are rabbit adipose mesenchymal stem cells.

[0011] Preferably, the sodium hyaluronate, the poloxamer-127 and the mesenchymal stem cell supernatant are mixed at 4 °C to obtain the hydrogel.

[0012] Preferably, the serum-free medium is a serum-free complete medium for mesenchymal stem cells;

[0013] The serum-free complete medium for mesenchymal stem cells is prepared from a mesenchymal stem cell additive, a serum-free basic medium for mesenchymal stem cells, and a broad-spectrum antibacterial agent in a volume ratio of 1-2:100-150:1-2, and the broad-spectrum antibacterial agent is a double antibody of penicillin-streptomycin.

[0014] Preferably, the method for obtaining the mesenchymal stem cell supernatant comprises the following steps:

[0015] Subculturing the isolated rabbit adipose mesenchymal stem cells with a serum-free complete medium for mesenchymal stem cells;

[0016] Culturing the rabbit adipose mesenchymal stem cells to passage 3, when the cell growth density reaches 80-90%, culturing with a low-sugar basic medium, collecting the supernatant, and obtaining the mesenchymal stem cell supernatant.

[0017] The hydrogel prepared by the described preparation method.

[0018] Use of the hydrogel in the preparation of a drug for promoting skin wound healing / trauma repair. The hydrophobic interaction of the three components can form spherical micelles in an aqueous solution. When the critical temperature and concentration are reached, these micelles closely aggregate to form a physically cross-linked gel structure, making the wound bind more tightly. Therefore, it can be used in the preparation of a drug for promoting skin wound healing / trauma repair. The drug is the hydrogel, and the preparation method of the drug comprises the following steps:

[0019] Cultivate mesenchymal stem cells with a serum-free medium to obtain a mesenchymal stem cell supernatant;

[0020] Mix the sodium hyaluronate, the poloxamer-127, and the mesenchymal stem cell supernatant at 4 °C to obtain the hydrogel;

[0021] The mass concentration of the sodium hyaluronate in every 100 mL of the mesenchymal stem cell supernatant is 0.3 g to 2.8 g;

[0022] The mass concentration of the poloxamer-127 in every 100 mL of the mesenchymal stem cell supernatant is 20 g to 30 g;

[0023] The serum-free medium is a serum-free complete mesenchymal stem cell medium;

[0024] The serum-free complete mesenchymal stem cell medium is prepared from a mesenchymal stem cell additive, a serum-free mesenchymal stem cell basal medium, and a broad-spectrum antibacterial agent with a volume ratio of 1:100:1;

[0025] The method for obtaining the mesenchymal stem cell supernatant comprises the following steps:

[0026] Subculture the isolated rabbit adipose mesenchymal stem cells with the serum-free complete mesenchymal stem cell medium;

[0027] Cultivate the rabbit adipose mesenchymal stem cells to passage 3. When the cell growth density reaches 80-90%, culture them with a low-glucose basal medium, collect the supernatant, and obtain the mesenchymal stem cell supernatant.

[0028] Use of the hydrogel in the preparation of a drug for promoting hair follicle growth. The drug for promoting hair follicle growth is the same as the drug for promoting skin wound healing / trauma repair.

[0029] Current research shows that injecting adipose mesenchymal stem cells or umbilical cord mesenchymal stem cells at multiple points is involved in the repair of skin tissue after trauma, and a variety of cytokines, exosomes, and small molecule substances secreted by them can promote the repair of epidermal and dermal tissues during the trauma process and inhibit excessive inflammatory reactions. However, there is no report on the study of preparing hydrogels by cross-linking PF-127 and sodium hyaluronate with the supernatant of adipose mesenchymal stem cells and treating skin injuries.

[0030] This gel has thermosensitivity. It is liquid at room temperature but can form a hydrogel at physiological temperature.

[0031] Poloxamer is a non-ionic surfactant copolymer composed of polyoxyethylene and polyoxypropylene. It is a biocompatible scaffold material approved by the US FDA for bioabsorbable use. It gels reversibly when the temperature rises and gradually dissolves in the body through simple surface absorption and clearance. Among them, Pluronic F-127, abbreviated as PF-127, has a molecular formula of PEO-PPO-PEO. The English name of polyoxyethylene is Polyoxtethylene, abbreviated as PEO, and the English name of polypropylene oxide is Polypropylene oxygen, abbreviated as PPO.

[0032] The inventive concept of the present invention lies in:

[0033] 1. As one of the main components of the extracellular matrix, sodium hyaluronate is widely distributed in animals and is rich in vitreous humor, synovial fluid, skin, nervous system, and umbilical cord, which can provide a potential method for wound dressings and skin repair. As a hydrogel scaffold for wound repair, PF-127 has characteristics such as injectability, biocompatibility, and thermosensitivity. The hydrophobic interaction of the three components can form spherical micelles in aqueous solution, and these micelles closely aggregate at the critical temperature and concentration to form a physically cross-linked gel structure, making the wound bond more tightly. Loading the cytokines in the supernatant onto the gel can prolong the residence time of the supernatant in the body.

[0034] 2. In order to simplify the procedures for purifying and identifying various cell products, avoid the hazards caused by virus contamination, reduce the adverse factors brought by serum, and make the cell culture conditions more stable, the present invention uses a serum-free medium for culturing adipose mesenchymal stem cells and loads the cytokines in the supernatant onto the gel to prolong the residence time of the supernatant at the wound site, providing a way similar to the controlled release of drugs.

[0035] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0036] The present invention demonstrates that a hydrogel loaded with mesenchymal stem cell supernatant is beneficial for wound healing and promotes wound closure, hair follicle and angiogenesis in vivo. Overall, the mesenchymal stem cell supernatant hydrogel represents a promising cell-free regenerative medicine approach for therapeutic and wound healing purposes. Description of the Drawings

[0037] Figure 1 It is a morphological result diagram of passage 3 mesenchymal stem cells under an inverted fluorescence microscope. Among them, A is a 5-fold magnification diagram, and B is a 10-fold magnification diagram.

[0038] Figure 2 It is a result diagram of the trilineage induction of mesenchymal stem cells. Among them, A is the result diagram of adipogenic induction, B is the result diagram of chondrogenic induction, and C is the result diagram of osteogenic induction.

[0039] Figure 3 It is a transwell migration diagram. Among them, A represents 20% PF-127 + supernatant, B represents 25% PF-127 + supernatant, C represents 30% PF-127 + supernatant, D represents 20% PF-127 + 0.3% HA + supernatant, and E represents 20% PF-127 + 2.8% HA + supernatant.

[0040] Figure 4 It is a thermosensitivity test diagram of the stem cell supernatant gel. Among them, A is in a liquid state at 4 degrees, and B is in a gel state at 37 degrees.

[0041] Figure 5 It is a healing effect diagram of the wounds of mice treated with different methods. Among them, A1 is the healing effect diagram of 20% PF-127 + 0.3% HA + supernatant on the first day, A2 is the healing effect diagram of 20% PF-127 + 0.3% HA + supernatant on the fifth day, A3 is the healing effect diagram of 20% PF-127 + 0.3% HA + supernatant on the eighth day, B1 is the healing effect diagram of PBS on the first day, B2 is the healing effect diagram of PBS on the fifth day, B3 is the healing effect diagram of PBS on the eighth day; C1 is the healing effect diagram of 20% PF-127 + supernatant on the first day, C2 is the healing effect diagram of 20% PF-127 + supernatant on the fifth day, C3 is the healing effect diagram of 20% PF-127 + supernatant on the eighth day, D1 is the healing effect diagram of 0.3% HA + supernatant on the first day, D2 is the healing effect diagram of 0.3% HA + supernatant on the fifth day, and D3 is the healing effect diagram of HA + supernatant on the eighth day.

[0042] Figure 6It is a HE staining section diagram for wound healing. Among them, A1 is the HE staining section diagram of 20% PF-127 + 0.3% HA + supernatant on the first day, A2 is the HE staining section diagram of 20% PF-127 + 0.3% HA + supernatant on the fifth day, A3 is the HE staining section diagram of 20% PF-127 + 0.3% HA + supernatant on the eighth day, B1 is the HE staining section diagram of PBS on the first day, B2 is the HE staining section diagram of PBS on the fifth day, B3 is the HE staining section diagram of PBS on the eighth day; C1 is the HE staining section diagram of 20% PF-127 + supernatant on the first day, C2 is the HE staining section diagram of 20% PF-127 + supernatant on the fifth day, C3 is the HE staining section diagram of 20% PF-127 + supernatant on the eighth day, D1 is the HE staining section diagram of 0.3% HA + supernatant on the first day, D2 is the HE staining section diagram of 0.3% HA + supernatant on the fifth day, D3 is the HE staining section diagram of 0.3% HA + supernatant on the eighth day. Note: NBV is new blood vessels; HF is hair follicles; F is fibroblasts.

[0043] Figure 7 It is the scanning imaging diagrams of each substance. Among them, A is the temperature-sensitive hydrogel loaded with mesenchymal stem cell supernatant in Example 1, B is PF-127, C is HA, D is 0.3% HA + supernatant, and E is 20% PF-127 + supernatant. Specific Embodiments

[0044] The specific embodiments of the present invention will be described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0045] The material information used in the present invention is as follows:

[0046] DPBS is purchased from Youkang, and the product number is BS0402;

[0047] Mesenchymal stem cell additive is purchased from Youkang, and the product number is NC0103;

[0048] Serum-free mesenchymal stem cell basal medium is purchased from Youkang, and the product number is NC0104.s;

[0049] Stem cell mild digestive enzyme is purchased from Youkang, and the product number is NC1004.2;

[0050] Adipose tissue digestive enzyme is purchased from gibco, and the product number is 17018029;

[0051] Low-glucose basal medium DMEM is purchased from gibco, and the product number is 8122462.

[0052] Preparation of serum-free mesenchymal stem cell complete culture medium

[0053] Completely thaw 5 mL of mesenchymal stem cell additive, add all of it to 500 mL of serum-free mesenchymal stem cell basal culture medium, and add 5 mL of broad-spectrum antibacterial agent, shake thoroughly to mix, the broad-spectrum antibacterial agent is a dual antibiotic of penicillin-streptomycin.

[0054] Preparation of rabbit adipose-derived mesenchymal stem cells (AD-MSCs)

[0055] 1. Isolation of primary AD-MSCs

[0056] Cut the adipose tissue into a paste-like shape and centrifuge at 1,300rpm for 5min. Add an equal volume of adipose tissue digestion enzyme to the tissue, and digest at 37°C for 30min to 60min. The digestion time in this embodiment is 50min. Add an equal volume of serum-free mesenchymal stem cell complete medium to the adipose tissue digestion enzyme to terminate the digestion, filter using an 80-mesh cell screen, centrifuge the filtrate at 1,300rpm for 5min, and remove the supernatant. Resuspend the cell pellet with serum-free mesenchymal stem cell complete medium to obtain P2 generation rabbit AD-MSCs, adjust the cell concentration, and inoculate the P2 generation rabbit AD-MSCs in a T25 culture flask and place it in an incubator at 37°C and 5% CO2 saturated humidity. After 48h of culture, the first fluid change was performed, and the fluid change was performed every 3d thereafter until the cell confluence reached 80% to 90% and then the culture was subcultured.

[0057] 2. Passaging of AD-MSCs

[0058] (1) When the P2 generation rabbit AD-MSCs occupy 80% to 90% of the bottom area of ​​the entire T25 culture flask, the original culture medium is discarded and the cells are washed with the pre-prepared DPBS buffer. After washing, the residual liquid in the flask is aspirated.

[0059] (2) Add 1 mL of pre-warmed stem cell mild digestion enzyme to the T25 culture flask in step (1) for digestion. The digestion time is controlled between 2 min and 5 min, and the longest time should not exceed 5 min. The digestion time in this embodiment is 3 min. When the adherent cells begin to shrink, become round, and begin to suspend under a microscope, gently tap the side wall of the culture flask to completely detach the cells.

[0060] (3) Add 2 mL of pre-warmed serum-free mesenchymal stem cell complete culture medium to the T25 culture flask in step (2) to terminate digestion, and gently blow the bottom of the culture flask to remove cells that have not completely detached from the wall.

[0061] (4) Aspirate the liquid in the culture flask into a 15 mL centrifuge tube, centrifuge using a centrifuge at 1,300 rpm for 5 min, discard the supernatant, and retain the cell pellet at the bottom of the tube.

[0062] (5) Resuspend with 1 mL of serum-free mesenchymal stem cell complete medium, gently pipette to mix evenly, disperse the aggregated cells, and do not pipette vigorously to prevent cell rupture.

[0063] (6) After dispersing and mixing evenly, aspirate 10 μL of the suspension, use a cell counter, and inoculate it into a new T25 flask at 5,000 cells / cm 2 for continuous passage culture.

[0064] 3. Preparation of rabbit adipose mesenchymal stem cell supernatant

[0065] Culture rabbit AD-MSCs to passage P3 until the cells reach 80% confluence. Replace the serum-free mesenchymal stem cell complete medium with low-glucose basal medium DMEM for culture. After culturing for 48 h, collect the supernatant in the culture flask and temporarily store it at 4°C. Centrifuge the obtained supernatant at 4,000 rpm for 30 min, retain the supernatant solution, remove cell debris, and then perform aseptic filtration of the supernatant solution using a 0.22 μM filter. The obtained filtrate is the available cell supernatant.

[0066] Example 1

[0067] Preparation of hydrogel loaded with mesenchymal stem cell supernatant

[0068] Work before hydrogel preparation:

[0069] 1. Preparation of PF-127 solution: Take 10 g, 15 g, 20 g, 25 g, and 30 g of PF-127 powder, respectively, place them in 100 mL of cell supernatant, stir evenly with a glass rod at 4°C, and after the powder is no longer visible, place it in a 4°C refrigerator to stand and remove bubbles overnight for use the next day.

[0070] 2. Preparation of sodium hyaluronate solution (HA): Take 2.8 g and 0.3 g of sodium hyaluronate powder, respectively, place them in 100 mL of cell supernatant, stir evenly, place them in a 37°C shaker for 30 min until the white powder is no longer visible, and place it in a 4°C refrigerator for standby.

[0071] 3. Perform thermosensitivity tests on five concentrations of PF-127 solutions and find that PF-127 with mass concentrations of 10% and 15% cannot form a gel at 37°C, so they are discarded.

[0072] Hydrogel preparation:

[0073] Mix 50 mL of cell supernatant with 0.3% (mass fraction) HA solution and 20% (mass fraction) PF-127 solution at 4 °C to obtain a hydrogel.

[0074] Example 2

[0075] The difference between Example 2 and Example 1 is that 50 mL of cell supernatant is mixed with 0.3% (mass fraction) HA solution and 25% (mass fraction) PF-127 solution at 4 °C to obtain a hydrogel.

[0076] Example 3

[0077] The difference between Example 3 and Example 1 is that 50 mL of cell supernatant is mixed with 0.3% (mass fraction) HA solution and 30% (mass fraction) PF-127 solution at 4 °C to obtain a hydrogel.

[0078] Comparative Example 1

[0079] Mix 50 mL of cell supernatant with 30% (mass fraction) PF-127 solution at 4 °C to obtain a hydrogel.

[0080] Comparative Example 2

[0081] Mix 50 mL of cell supernatant with 20% (mass fraction) PF-127 solution at 4 °C to obtain a hydrogel.

[0082] Comparative Example 3

[0083] Mix 50 mL of cell supernatant with 25% (mass fraction) PF-127 solution at 4 °C to obtain a hydrogel.

[0084] Comparative Example 4

[0085] Mix 50 mL of cell supernatant with 2.8% (mass fraction) HA solution at 4 °C to obtain a hydrogel.

[0086] Comparative Example 5

[0087] Mix 50 mL of cell supernatant with 0.3% (mass fraction) HA solution at 4 °C to obtain a hydrogel.

[0088] Comparative Example 6

[0089] Mix 50 mL of cell supernatant with 2.8% (mass fraction) HA solution and 20% (mass fraction) PF-127 solution at 4 °C to obtain a hydrogel.

[0090] Effect Verification

[0091] 1. Invert the obtained passage 3 mesenchymal stem cells and observe them under a fluorescence microscope. The morphological results are as Figure 1 shown, from Figure 1It can be seen that after 8 - 12 days of culture, under an inverted fluorescence microscope, the cell growth confluence reached 80%, and the cells showed a typical polar vortex-like growth with a single and uniform morphology, proving that mesenchymal stem cells were successfully extracted and could be used for subsequent experiments.

[0092] 2. The obtained mesenchymal stem cells were induced in three lineages, and the results were as Figure 2 shown. As Figure 2 can be seen, after 21 days of induction with the adipogenic differentiation induction medium, most of the cell morphologies gradually changed into flat circles, the cell volume increased, and many lipid droplets appeared. After the induced cells were stained with Oil Red O and observed under a microscope, as Figure 2 shown, it can be seen that the lipid droplets in the cytoplasm of AD-MSCs were stained red. After 21 days of three-dimensional induction culture with the chondrogenic differentiation induction medium, the chondrospheres were fixed with formalin and embedded in paraffin for sectioning, and stained with Alcian blue. It can be seen under a microscope that the acidic mucopolysaccharides in the cartilage tissue were stained blue, indicating that the mesenchymal stem cells isolated and cultured without serum had good proliferation ability and adipogenic, osteogenic, and chondrogenic differentiation abilities.

[0093] 3. The prepared hydrogel was placed in the upper chamber of the transwell in a 24-well plate at 37°C, and 100 μL of dPBS was placed in the lower layer of cells. The liquid in the lower chamber was taken at 24 h, 48 h, and 72 h respectively, and the BCA method was used to quantify the protein concentration in the lower chamber and calculate the percentage of the released protein. The results are shown in Table 1 and Figure 3 , and Table 1 shows the migration rates of the three components of PF-127, HA, and the supernatant, Figure 3 reflecting the migration rate trend and significance.

[0094] As Figure 3 can be seen, at different times, the migration of mesenchymal stem cell supernatant among different concentrations of thermosensitive gels was observed. The migration rate was the lowest at the concentration of 20% PF-127 + 2.8% HA. At the concentration of 20% PF-127 + 0.3% HA, the migration rate was the highest and most persistent, with sustained release. Compared with other groups, the migration rate of the thermosensitive hydrogel of the stem cell supernatant at the concentration of 20% PF-127 + 0.3% HA was the highest, enabling cytokines to stay at the wound surface for a long time, which was beneficial to promoting the tight binding of the wound. And as time went by, the migration rates of 25% PF-127, 30% PF-127, and 20% PF-127 + 2.8% HA all decreased. Therefore, the optimal concentration was 20% PF-127 + 0.3% HA.

[0095] Table 1 Migration rates of different components

[0096]

[0097] 4. Thermosensitivity test:Figure 4 This is a graph showing the thermosensitivity test of the stem cell supernatant gel. At 4°C, the gel is in a liquid state. When the gel is inverted at 37°C, the gel becomes solidified. Therefore, this gel has thermosensitivity.

[0098] 5. In vivo experiment: 36 mice were divided into four groups, namely Group A, Group B, Group C, and Group D. A skin injury model was established on the back of each mouse. Using a sterile surgical scissor, a skin tissue with a diameter of 10 mm was cut off from the back. Medication started on the same day. Group A was the PF-127 + HA + supernatant treatment group, three times a day, 1 mL each time. Group B was the PBS control group, three times a day, 1 mL each time. Group C was the PF-127 + supernatant group, three times a day, 1 mL each time. Group D was the HA + supernatant group, three times a day, 1 mL each time. The mice were sacrificed on the 8th day. Photos were taken and HE staining sections were prepared on the 1st, 5th, and 8th days to observe the recovery situation.

[0099] Figure 5 This is a graph showing the healing effect of different treatment methods on the mouse wound surface. As can be seen from the graph, the skin injuries in each group have all recovered to some extent. However, on the fifth day, a scab formed on the skin surface of the mice in Group A, and the recovery was the fastest. The thermosensitive hydrogel loaded with the supernatant of mesenchymal stem cells in Group A had the best effect.

[0100] The healing rates of different treatment methods on the mouse wound surface are shown in Table 2. As can be seen from Table 2, there were slight differences in the healing rates of the skin injuries in each group on the first day and the eighth day. On the fifth day, the skin healing rate of the PF-127 + HA + supernatant group was higher than that of other groups. Compared with other groups, the PF-127 + HA + supernatant group had the highest healing rate. This proves that the thermosensitive hydrogel loaded with the supernatant of mesenchymal stem cells at this concentration has the best effect.

[0101] Table 2 Healing rates of different treatment methods on the mouse wound surface

[0102]

[0103] Figure 6 This is a graph of the HE staining section of wound healing. As can be seen from the graph, there were no obvious differences in each group on the first day. On the fifth day, new blood vessels grew in Group A and Group B. Among them, the number of fibroblasts in Group A increased, promoting wound recovery. Compared with other groups, the wounds treated with the treatment group showed increased re-epithelialization starting from the wound edge, significantly increasing the number of fibroblasts, angiogenesis, and the number of hair follicles, which significantly promoted the healing process. On the eighth day, many hair follicles and new blood vessels grew in Group A.

[0104] On the 5th day, paraffin section observation showed that obvious changes had begun to appear in the morphological structure of tissue cells at the injury site, and the degree of tissue necrosis and apoptosis was slight. The skin lesions in group A healed better, and new skin had formed at the wound edge, with a small amount of fibrous tissue formed. However, the skin lesions in group B healed poorly, and no new skin had formed at the wound edge, only a large amount of necrotic tissue and inflammatory cell infiltration. On the 8th day, paraffin section observation showed that the morphological structure of tissue cells at the injury site had basically returned to normal, and the degree of tissue necrosis and apoptosis was relatively light. In group A, more new skin had formed at the wound edge, and more fibrous tissue, as well as the structures of tiny hair follicles and sebaceous glands, had formed, indicating that the skin wound area and wound healing rate of the thermosensitive hydrogel loaded with the supernatant of mesenchymal stem cells at the concentration of group A were better than those of other groups.

[0105] On the 5th day after injury, the number of inflammatory cells in the treatment group decreased. Compared with other groups, the wound treated with the treatment group started re-epithelialization from the wound edge, increased the number of fibroblasts, increased angiogenesis and the number of hair follicles, and significantly promoted the healing process.

[0106] Figure 7 As shown in the scanning electron microscope image of the thermosensitive hydrogel loaded with the supernatant of mesenchymal stem cells, it can be seen from the figure that this hydrogel has a highly porous network structure, forming a crosslinked gel structure, which is loose and porous, facilitating the components in the supernatant of the loaded mesenchymal stem cells to bind to a variety of cytokines, enabling better slow and long-term release of active substances, and promoting the tight combination of the wound.

[0107] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that any value between the two endpoints of each numerical range and the two endpoints themselves can be selected. To avoid redundancy, the preferred embodiments of the present invention are described.

[0108] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0109] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing a hydrogel loaded with mesenchymal stem cell supernatant, characterized in that: The following steps are involved: Cultivating mesenchymal stem cells with a serum-free culture medium to obtain a mesenchymal stem cell supernatant; Mixing the mesenchymal stem cell supernatant with sodium hyaluronate and poloxamer-127 to obtain the hydrogel; The mass concentration of the sodium hyaluronate in each 100 mL of the mesenchymal stem cell supernatant is 0.3 g; The mass concentration of poloxamer-127 in every 100 mL of the mesenchymal stem cell supernatant is 20 g to 30 g.

2. The preparation method according to claim 1, characterized in that: The mesenchymal stem cells are rabbit adipose mesenchymal stem cells.

3. The preparation method according to claim 1, characterized in that: The sodium hyaluronate, the poloxamer-127 and the mesenchymal stem cell supernatant were mixed at 4° C. to obtain the hydrogel.

4. The preparation method according to claim 1, characterized in that: The serum-free culture medium is a serum-free mesenchymal stem cell complete culture medium; The serum-free mesenchymal stem cell complete culture medium is prepared from a mesenchymal stem cell additive, a serum-free mesenchymal stem cell basal culture medium, and a broad-spectrum antibacterial agent in a volume ratio of 1-2:100-150:1-2.

5. The preparation method according to claim 4, characterized in that: The method for obtaining the mesenchymal stem cell supernatant comprises the following steps: The isolated rabbit adipose-derived mesenchymal stem cells were subcultured with serum-free mesenchymal stem cell complete medium; Rabbit adipose mesenchymal stem cells were cultured to P3 generation. When the cell growth density reached 80-90%, they were cultured in a low-glucose basal medium, and the supernatant was collected to obtain mesenchymal stem cell supernatant.

6. The hydrogel prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the hydrogel according to claim 6 in the preparation of a drug for promoting skin wound healing / wound repair.

8. Use of the hydrogel according to claim 6 in preparing a drug for promoting hair follicle growth.

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