Hydrogel loaded with serum-free culture mesenchymal stem cell supernate as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological gel, and particularly relates to hydrogel loaded with serum-free culture mesenchymal stem cell supernate and a preparation method and application of the hydrogel. Sodium hyaluronate, the mesenchymal stem cell supernate and poloxamer-127 are evenly mixed to prepare the hydrogel. The mass concentration of the sodium hyaluronate in every 100 mL of the mesenchymal stem cell supernatant is 0.3-2 g, and the mass concentration of the poloxamer-127 is 20-30 g. When the adipose-derived mesenchymal stem cells are cultured, a serum-free culture medium is adopted for culture, and cell factors in the supernatant are loaded on the gel, so that the retention time of the supernatant is prolonged.

Description

Technical Field

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

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

[0003] Mesenchymal stem cells come from the mesoderm and can differentiate into various cell types such as osteoblasts, chondrocytes and adipocytes. After in vitro expansion, they can treat autoimmune diseases such as psoriasis, osteoarthritis, anal fistula, amyotrophic lateral sclerosis and other diseases. Its cell supernatant contains a large number of cytokines, including proteins such as COL1A1, COL1A2, FN1, TNC, COL4A, which are proteins that play an important role in the wound recovery process. The cells are cultured continuously in vitro and then decellularized, 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, promote the proliferation of fibroblasts, and use the rich extracellular matrix secreted by the cells to form an endogenous scaffold, thereby avoiding the various disadvantages of exogenous scaffolds.

[0004] Generally, serum is required to culture animal mesenchymal stem cells. Without serum, most cells cannot proliferate. Currently, fetal bovine serum is commonly used as serum. Studies have found that it has the risk of contamination with exogenous viruses and pathogenic factors, which can easily cause infection to the wound surface, and cannot solve the problem that the cytokines of mesenchymal stem cells stay in the wound for a short time. Summary of the invention

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

[0006] A method for preparing a hydrogel loaded with mesenchymal stem cell supernatant comprises the following steps:

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

[0008] The mesenchymal stem cell supernatant is mixed 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 0.3g to 2.8g; the mass concentration of poloxamer-127 in each 100 mL of the mesenchymal stem cell supernatant is 20g to 30g. When culturing adipose mesenchymal stem cells, the present invention uses a serum-free medium for culturing to avoid infection, and loads cytokines in the supernatant onto the gel to prolong the residence time of the supernatant in the body.

[0009] Preferably, the mass concentration of the 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 culture medium is a serum-free mesenchymal stem cell complete culture medium;

[0013] 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, and the broad-spectrum antibacterial agent is a dual antibiotic of penicillin-streptomycin.

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

[0015] The isolated rabbit adipose-derived mesenchymal stem cells were subcultured with serum-free mesenchymal stem cell complete medium;

[0016] Rabbit adipose mesenchymal stem cells are cultured to P3 generation. When the cell growth density reaches 80-90%, low-sugar basal medium is used for culture, and supernatant is collected to obtain mesenchymal stem cell supernatant.

[0017] The hydrogel prepared by the preparation method.

[0018] The hydrogel is used in the preparation of a drug for promoting skin wound healing / wound 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 are tightly aggregated to form a physically cross-linked gel structure, making the wound more tightly bonded. Therefore, it can be used to prepare a drug for promoting skin wound healing / wound repair. The drug is the hydrogel, and the preparation method of the drug comprises the following steps:

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

[0020] Mixing 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 each 100 mL of the mesenchymal stem cell supernatant is 0.3 g to 2.8 g;

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

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

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

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

[0026] The isolated rabbit adipose-derived mesenchymal stem cells were subcultured with serum-free mesenchymal stem cell complete medium;

[0027] Rabbit adipose mesenchymal stem cells are cultured to P3 generation. When the cell growth density reaches 80-90%, low-sugar basal medium is used for culture, and supernatant is collected to obtain mesenchymal stem cell supernatant.

[0028] The hydrogel is used 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 / wound repair.

[0029] At present, studies have shown that point-by-point injection of adipose-derived mesenchymal stem cells or umbilical cord-derived mesenchymal stem cells is involved in the repair of skin tissue after trauma, and the various cytokines, exosomes and small molecules secreted by them can promote the repair of epidermal and dermal tissues during trauma and inhibit excessive inflammatory responses. However, there have been no reports on the use of adipose-derived mesenchymal stem cell supernatant to cross-link PF-127 and sodium hyaluronate to prepare hydrogels and treat skin injuries.

[0030] The gel is temperature-sensitive and is liquid at room temperature, but can form a hydrogel at physiological temperature.

[0031] Poloxamer is a nonionic surface active copolymer composed of polyoxyethylene and polyoxypropylene. It is a bioabsorbable biocompatible stent material approved by the US FDA. It reversibly gels when the temperature rises and gradually dissolves in the body through simple surface absorption and removal. Among them, PluronicF-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 polyoxypropylene is Polypropylene oxygen, abbreviated as PPO.

[0032] The inventive concept of the present invention is:

[0033] 1. Sodium hyaluronate, as one of the main components of the extracellular matrix, is widely distributed in animals and is abundant in the vitreous, joint fluid, skin, nervous system and umbilical cord. It can provide a potential method for wound dressings and skin repair. PF-127, as a hydrogel scaffold for wound repair, has the characteristics of injectability, biocompatibility and thermosensitivity. The hydrophobic interaction of the three components can form spherical micelles in aqueous solution. When the critical temperature and concentration are reached, these micelles are tightly aggregated to form a physically cross-linked gel structure, making the wound more tightly bound. The cytokines in the supernatant are loaded onto the gel to 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 harm caused by virus contamination, reduce the adverse factors brought by serum, and make the cell culture conditions more stable, the present invention uses serum-free culture medium for culturing adipose-derived mesenchymal stem cells, and loads cytokines in the supernatant onto the gel to prolong the residence time of the supernatant at the wound, providing a method similar to drug controlled release.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention demonstrates that hydrogels loaded with MSC supernatant are beneficial for wound healing and promote wound closure, hair follicle and angiogenesis in vivo. Overall, MSC supernatant hydrogels represent a promising cell-free regenerative medicine approach for therapeutic and wound healing purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 These are the morphological results of P3 mesenchymal stem cells under an inverted fluorescence microscope, where A is a 5-fold magnification and B is a 10-fold magnification.

[0038] Figure 2 These are the results of three-lineage induction of mesenchymal stem cells, where A is the result of adipogenic induction, B is the result of chondrogenic induction, and C is the result of osteogenic induction.

[0039] Figure 3 This is a transwell migration diagram, in which 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 This is a test diagram of the temperature sensitivity of stem cell supernatant gel, where A is liquid at 4 degrees and B is gel at 37 degrees.

[0041] Figure 5 The figures are the healing effect diagrams of different treatment methods on mouse wounds, among which, 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, and 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 6HE staining sections of wound healing, wherein A1 is a HE staining section of 20% PF-127+0.3% HA+supernatant on the first day, A2 is a HE staining section of 20% PF-127+0.3% HA+supernatant on the fifth day, A3 is a HE staining section of 20% PF-127+0.3% HA+supernatant on the eighth day, B1 is a HE staining section of PBS on the first day, B2 is a HE staining section of PBS on the fifth day, and B3 is a HE staining section of PBS on the eighth day; C1 is The first day is the HE-stained section with 20% PF-127+supernatant, C2 is the HE-stained section with 20% PF-127+supernatant on the fifth day, C3 is the HE-stained section with 20% PF-127+supernatant on the eighth day, D1 is the HE-stained section with 0.3% HA+supernatant on the first day, D2 is the HE-stained section with 0.3% HA+supernatant on the fifth day, D3 is the HE-stained section with 0.3% HA+supernatant on the eighth day. Note: NBV is new blood vessel; HF is hair follicle; F is fibroblast.

[0043] Figure 7 The scanned images of the substances are shown in Figure 1, wherein A is the thermosensitive hydrogel loaded with the supernatant of mesenchymal stem cells in Example 1, B is PF-127, C is HA, D is 0.3% HA+supernatant, and E is 20% PF-127+supernatant. DETAILED DESCRIPTION

[0044] The specific embodiments of the present invention are 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 ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

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

[0046] DPBS was purchased from Youkang, catalog number BS0402;

[0047] Mesenchymal stem cell supplement was purchased from Youkang, catalog number NC0103;

[0048] Serum-free mesenchymal stem cell basal medium was purchased from Youkang, catalog number NC0104.s;

[0049] Stem cell mild digestive enzyme was purchased from Youkang, catalog number NC1004.2;

[0050] Adipose tissue digestive enzymes were purchased from Gibco, catalog number 17018029;

[0051] The low-glucose basal medium DMEM was purchased from Gibco with the catalog number 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) Pipette the liquid in the culture flask into a 15 mL centrifuge tube and 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 the cells in 1 mL of serum-free mesenchymal stem cell complete medium and gently pipette to mix to disperse the aggregated cells. Do not pipette violently to prevent cell rupture.

[0063] (6) After blowing and mixing, aspirate 10 μL of the suspension and use a cell counter to count at 5,000 cells / cm 2 Inoculate into new T25 flasks and continue subculture.

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

[0065] Rabbit AD-MSCs were cultured to P3 until the cells reached 80% confluence. The serum-free mesenchymal stem cell complete medium was replaced with a low-glucose basal medium DMEM for culture. After 48 hours of culture, the supernatant in the culture flask was collected and temporarily stored at 4°C. The obtained supernatant was centrifuged at 4,000 rpm for 30 minutes, the supernatant solution was retained, the cell debris was removed, and then the supernatant solution was sterile filtered using a 0.22 μM filter, and the filtrate obtained was the usable cell supernatant.

[0066] Example 1

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

[0068] Preparing hydrogels:

[0069] 1. Preparation of PF-127 solution: Take 10g, 15g, 20g, 25g, and 30g of PF-127 powder and put them into 100mL of cell supernatant respectively. Stir evenly with a glass rod at 4℃. When the powder is no longer visible, put it in a 4℃ refrigerator to stand and remove bubbles overnight. Set it aside the next day.

[0070] 2. Preparation of sodium hyaluronate solution (HA): Take 2.8g and 0.3g of sodium hyaluronate powder, respectively, put them into 100mL of cell supernatant and mix them evenly. Put them into a 37℃ shaking pot and shake for 30min until no white powder is visible. Put them into a 4℃ refrigerator for later use.

[0071] 3. The temperature sensitivity of PF-127 solutions with five concentrations was tested. It was found that PF-127 with a mass concentration of 10% and 15% could not form a gel at 37°C, so they were discarded.

[0072] Hydrogel preparation:

[0073] 50 mL of cell supernatant was mixed with 0.3% by mass HA solution and 20% by mass 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, 0.3% by mass HA solution and 25% by mass PF-127 solution are mixed 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, 0.3% by mass HA solution and 30% by mass PF-127 solution are mixed at 4° C. to obtain a hydrogel.

[0078] Comparative Example 1

[0079] 50 mL of cell supernatant was mixed with 30% by mass PF-127 solution at 4° C. to obtain a hydrogel.

[0080] Comparative Example 2

[0081] 50 mL of cell supernatant was mixed with 20% by mass PF-127 solution at 4° C. to obtain a hydrogel.

[0082] Comparative Example 3

[0083] 50 mL of cell supernatant was mixed with 25% by mass PF-127 solution at 4° C. to obtain a hydrogel.

[0084] Comparative Example 4

[0085] 50 mL of cell supernatant was mixed with 2.8% HA solution at 4° C. to obtain a hydrogel.

[0086] Comparative Example 5

[0087] 50 mL of cell supernatant was mixed with 0.3% HA solution at 4° C. to obtain a hydrogel.

[0088] Comparative Example 6

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

[0090] Effect verification

[0091] 1. The obtained P3 mesenchymal stem cells were inverted and observed under a fluorescence microscope. The morphological results were as follows: Figure 1 As shown by 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 typical polar vortex growth with a single and uniform morphology, proving that mesenchymal stem cells were successfully extracted and can be used for subsequent experiments.

[0092] 2. The obtained mesenchymal stem cells were induced into three lines. The results are as follows Figure 2 As shown by Figure 2 It can be seen that after 21 days of induction of MSCs with adipogenic differentiation induction solution, most of the cells gradually changed into flat round shapes, the cell volume increased, and many lipid droplets appeared. After the induced cells were stained with Oil Red O and observed under a microscope, Figure 2 As shown, the lipid droplets in the cytoplasm of AD-MSCs were stained red. After MSCs were cultured in a chondrogenic differentiation induction medium for 21 days, the chondrocytes were fixed in formalin, embedded in paraffin, and sectioned. They were stained with Alecin blue. Under a microscope, it was observed that the acidic mucopolysaccharide in the cartilage tissue was stained blue, indicating that the mesenchymal stem cells separated and cultured in serum-free conditions had good proliferation ability and differentiation ability into adipogenesis, osteogenesis, and chondrogenesis.

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

[0094] Depend on Figure 3 It can be seen that at different times, the migration of mesenchymal stem cell supernatant between thermosensitive gels of different concentrations, 20% PF-127 + 2.8% HA concentration has the lowest migration rate. At 20% PF-127 + 0.3% HA concentration, the migration rate is the highest and most lasting, with sustained release. Compared with other groups, 20% PF-127 + 0.3% HA concentration stem cell supernatant thermosensitive hydrogel has the highest migration rate, allowing cytokines to stay in the wound for a long time, which is conducive to promoting the close bonding of the wound, and over time, the migration rates of 25% PF-127, 30% PF-127, and 20% PF-127 + 2.8% HA all decreased. Therefore, the optimal concentration is 20% PF-127 + 0.3% HA.

[0095] Table 1 Migration of different components

[0096]

[0097] 4. Temperature sensitivity test: Figure 4 This is a test chart of the temperature sensitivity of stem cell supernatant gel. At 4 degrees, the gel is liquid. At 37 degrees, when the gel is inverted, the gel is solidified, so this gel is temperature sensitive.

[0098] 5. In vivo experiment: 36 mice were divided into four groups: A, B, C, and D. A skin injury model was established on the back of each mouse. The skin tissue with a diameter of 10 mm was cut off on the back with sterile surgical scissors. The medication was 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 killed on the 8th day. Photos and HE-stained sections were taken on the 1st, 5th, and 8th days to observe the recovery.

[0099] Figure 5 This is a graph showing the healing effects of different treatments on mouse wounds. As can be seen from the figure, the skin damage in each group was restored, but the skin of mice in Group A formed scabs on the fifth day and recovered the fastest. The thermosensitive hydrogel loaded with mesenchymal stem cell supernatant in Group A had the best effect.

[0100] The healing rates of mouse wounds treated with different treatments are shown in Table 2. It can be seen from Table 2 that there are slight differences in the healing rates of skin injuries in each group on the first and eighth days. On the fifth day, the skin healing rate of the PF-127+HA+supernatant group is higher than that of other groups. Compared with other groups, the healing rate of the PF-127+HA+supernatant group is the highest, which proves that the thermosensitive hydrogel loaded with this concentration of mesenchymal stem cell supernatant has the best effect.

[0101] Table 2 The healing rate of mouse wounds by different treatments

[0102]

[0103] Figure 6 This is a HE-stained section of wound healing. It can be seen from the figure that there was no significant difference between the groups on the first day. On the fifth day, new blood vessels grew in groups A and B, and the number of fibroblasts in group A increased, which promoted wound recovery. Compared with other groups, the wounds treated in the treatment group re-epithelialized from the wound edge, increased the number of fibroblasts, increased angiogenesis and the number of hair follicles, and 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 the morphology of tissue cells at the site of injury had begun to change significantly, and the degree of tissue necrosis and apoptosis was mild. The skin lesions in group A healed better, with new skin formed at the edge of the wound and a small amount of fibrous tissue formed. The skin lesions in group B healed poorly, with no new skin formed at the edge of the wound, only a large amount of necrotic tissue and inflammatory cell infiltration. On the 8th day, paraffin section observation showed that the morphology of tissue cells at the site of injury had basically returned to normal, and the degree of tissue necrosis and apoptosis was mild. There was more new skin formed at the edge of the wound in group A, and more fibrous tissue and tiny hair follicles and sebaceous glands were formed, indicating that the skin wound area and wound healing speed 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] The number of inflammatory cells in the treatment group decreased. On the 5th day after injury, the wounds treated in the treatment group re-epithelialized from the wound edge compared with the other groups, increasing the number of fibroblasts and angiogenesis and the number of hair follicles, which significantly promoted the healing process.

[0106] Figure 7 This is an image of a thermosensitive hydrogel loaded with mesenchymal stem cell supernatant under a scanning electron microscope. It can be seen from the figure that this hydrogel has a highly porous network structure, forming a cross-linked gel structure. The loose and porous structure is conducive to loading the components in the mesenchymal stem cell supernatant. It can combine with a variety of cytokines, and can better achieve the slow and long-term release of active substances, thereby promoting close binding of the wound.

[0107] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0108] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall 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 equivalents, 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 to 2 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 mass concentration of the sodium hyaluronate in every 100 mL of the mesenchymal stem cell supernatant is 0.3 g.

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

4. 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.

5. 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.

6. The preparation method according to claim 5, 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 are cultured to P3 generation. When the cell growth density reaches 80-90%, low-sugar basal medium is used for culture, and supernatant is collected to obtain mesenchymal stem cell supernatant.

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

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

9. Use of the hydrogel according to claim 7 in preparing a drug for promoting hair follicle growth.

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