Stem cell and its use in the preparation of products with biological tissue repair action

Abstract

This invention discloses the application of stem cells in the preparation of products with biological tissue repair functions, relating to the field of tissue engineering technology. The method includes the following steps: injecting an adipose-derived stem cell suspension onto a biological scaffold, culturing and proliferating it to obtain a product with biological tissue repair functions; the biological scaffold is made of hyaluronic acid-modified chitosan-polycaprolactone. The biological scaffold of this invention has high porosity and high strength, providing appropriate biomechanical support and a biochemical environment for cell adhesion, migration, proliferation, and osteogenic differentiation, thereby improving tissue repair functions, and it also exhibits excellent biocompatibility.

Description

Technical Field

[0001] This invention relates to the field of tissue engineering technology, specifically to a stem cell and its application in the preparation of products with biological tissue repair functions. Background Technology

[0002] Biomaterials can influence intercellular communication between adjacent cells and accelerate tissue regeneration. Scaffolds, as carriers of Exos and biological factors, greatly influence the activity of various factors and provide appropriate biomechanical support and biochemical environment for cell adhesion, migration, proliferation, and differentiation.

[0003] CN108187142A discloses an application of stem cells in biological tissue repair. It utilizes induced differentiated adipose stem cells to proliferate on a three-dimensional biomaterial scaffold to accelerate the healing of damaged skin, shorten the healing time, make the wound surface smoother, reduce skin wrinkles, and achieve a beautifying effect after skin healing. It provides a better healing condition for skin growth and recovery and provides a reference for the clinical treatment of skin trauma using stem cells. However, its scaffold porosity is insufficient, making it difficult to provide appropriate biomechanical support and biochemical environment for cells. Summary of the Invention

[0004] This invention provides a stem cell and its application in the preparation of products with biological tissue repair effects, exhibiting excellent tissue repair properties.

[0005] The present invention solves its technical problem by adopting the following technical solution:

[0006] The application of stem cells in the preparation of products with biological tissue repair functions includes the following steps:

[0007] Adipose-derived stem cell suspension is injected onto a biological scaffold, cultured and proliferated to obtain a product with biological tissue repair function;

[0008] The bioscaffold is made of hyaluronic acid-modified chitosan-polycaprolactone.

[0009] The biological scaffold of the present invention has high porosity and high strength, which can provide appropriate biomechanical support and biochemical environment for cell adhesion, migration, proliferation and osteogenic differentiation, thereby improving tissue repair, and it has excellent biocompatibility.

[0010] As a preferred embodiment of the present invention, the method for preparing the adipose stem cell suspension includes the following steps:

[0011] Adipose tissue was taken from the subcutaneous groin area of ​​SD rats, rinsed, minced, digested, centrifuged, and resuspended in PBS to obtain a preliminary cell suspension.

[0012] The cell suspension was placed in culture medium and cultured for 2-4 days. The medium was changed to remove non-adherent cells. After culturing for another 4-8 days, P1 generation adipose-derived stem cells were obtained.

[0013] P1 generation adipose-derived stem cells were passaged to the 5th generation to obtain adipose-derived stem cell suspension.

[0014] This invention obtains adipose-derived stem cell suspensions with excellent differentiation and abundant cell number per unit volume by rinsing, mincing, digesting, centrifuging, and then culturing adipose tissue from the subcutaneous groin area of ​​SD rats. The cell yield and cell viability are high, and the cells can be massively expanded and have a high survival rate after being seeded onto a biological scaffold.

[0015] In a preferred embodiment of the present invention, the rinsing is performed using Hanks solution; and / or

[0016] The digestion process utilizes type II collagenase.

[0017] In a preferred embodiment of the present invention, the culture medium comprises the following components: 60-100 mL / L inactivated bovine serum, 600-1000 IU / mL interleukin-5, 1-5 g / L Ganoderma lucidum polysaccharide, 1-5 g / L genistein, 10-100 mg / L glutathione, 20-100 mg / L L-galacturonic acid, 10-50 mg / L rhamnose galacturonic acid, 0.1-1 mg / L acetaminophen, 1-10 mg / L basic fibroblast growth factor, 1-10 mg / L TGF-β, 100-500 mg / L HEPES, 20-200 mL / L FBS, and the balance being DEME medium. In particular, the culture medium of the present invention results in high cell yield and high cell viability.

[0018] In a preferred embodiment of the present invention, the culture medium comprises the following components: 80 mL / L inactivated bovine serum, 800 IU / mL interleukin-5, 2 g / L Ganoderma lucidum polysaccharide, 3 g / L genistein, 40 mg / L glutathione, 80 mg / L L-galacturonic acid, 40 mg / L rhamnose galacturonic acid, 0.5 mg / L acetaminophen, 8 mg / L basic fibroblast growth factor, 8 mg / L TGF-β, 400 mg / L HEPES, 50 mL / L FBS, and the remainder being DEME medium. In particular, the culture medium with these amounts of raw materials results in high cell yield and high cell viability.

[0019] In a preferred embodiment of the present invention, the culture is carried out at 35°C and 5% CO2 concentration.

[0020] As a preferred embodiment of the present invention, the method for preparing the biological scaffold includes the following steps:

[0021] (1) Hyaluronic acid is added to water to prepare a hyaluronic acid solution with a concentration of 5-20%. Modified chitosan is added to water, dispersed evenly, and ultrasonically treated to obtain the precursor.

[0022] (2) Dissolve polycaprolactone in chloroform to prepare a polycaprolactone solution with a concentration of 15-25%, add the precursor, stir evenly, add sodium chloride, stir evenly, degas by ultrasonication, inject into a polytetrafluoroethylene mold, let stand for 36-48 hours, then demold, let stand for another 18-36 hours, and finally vacuum dry for 24-36 hours to obtain a biological scaffold.

[0023] In a preferred embodiment of the present invention, the method for preparing the biological scaffold comprises a hyaluronic acid to modified chitosan mass ratio of 1:(2~5); and / or

[0024] The mass ratio of polycaprolactone to the precursor is (70~85):(15~30).

[0025] As a preferred embodiment of the present invention, the method for preparing the modified chitosan includes the following steps:

[0026] Chitosan was subjected to plasma treatment to obtain pretreated chitosan;

[0027] 3-glycidyl etheroxypropyltriethoxysilane and phenyltris(dimethylsiloxane)silane were added to acetone and mixed evenly to obtain the modified solution.

[0028] Pretreated chitosan and calcium 3-hydroxy-3-methylbutyrate were added to the modification solution, stirred evenly in a water bath at 55~80℃, ultrasonically treated, filtered, and dried to obtain modified chitosan.

[0029] In a preferred embodiment of the present invention, the plasma treatment power is 400~1200W, and the time is 0.5~5min; and / or

[0030] The mass ratio of the pretreated chitosan, calcium 3-hydroxy-3-methylbutyrate, 3-glycidyl etheroxypropyltriethoxysilane, and phenyltris(dimethylsiloxane)silane is 1:(0.1~0.4):(0.01~0.05):(0.01~0.05).

[0031] This invention creatively modifies chitosan and adds calcium 3-hydroxy-3-methylbutyrate, which improves the scaffold's compatibility and porosity, enabling it to provide better biomechanical support and biochemical environment for cell adhesion, migration, proliferation, and osteogenic differentiation.

[0032] The beneficial effects of the present invention are as follows: (1) The biological scaffold of the present invention has high porosity and high strength, which can provide appropriate biomechanical support and biochemical environment for cell adhesion, migration, proliferation and osteogenic differentiation, thereby improving tissue repair and having excellent biocompatibility; (2) The present invention obtains adipose stem cell suspension with excellent differentiation degree and abundant cell number per unit volume by rinsing, cutting, digesting, centrifuging and culturing the adipose tissue in the subcutaneous groin of SD rats. The cell yield is high and the cell viability is high. After being seeded into the biological scaffold, it can be expanded in large quantities and has a high survival rate. Attached Figure Description

[0033] Figure 1 The image shown is a SEM image of the modified chitosan prepared in Example 1. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0036] In this application, numerical ranges are referred to as continuous unless otherwise specified, and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0037] In this application, there are no particular restrictions on the specific dispersion and mixing methods.

[0038] Unless otherwise specified, all reagents or instruments used in this application are commercially available products.

[0039] In this invention, all parts are by weight.

[0040] Example 1

[0041] The application of stem cells in the preparation of a product with biological tissue repair function, wherein the product with biological tissue repair function (adipose-derived stem cells combined with a biological scaffold) includes the following steps:

[0042] (1) Preparation of adipose-derived stem cell suspension:

[0043] Adipose tissue was taken from the subcutaneous groin area of ​​SD rats, rinsed with Hanks' solution, and then minced with scissors to a volume of 1 mm. 3 Add 0.16% type II collagenase to digest for 30 min, centrifuge at 3000 rpm for 10 min, resuspend the precipitate in PBS to obtain the initial cell suspension;

[0044] The initial cell suspension was placed in culture medium and cultured for 3 days. The medium was changed to remove non-adherent cells. After culturing for another 7 days, P1 generation adipose-derived stem cells were obtained.

[0045] P1 generation adipose-derived stem cells were passaged to the 5th generation to obtain adipose-derived stem cell suspension.

[0046] The culture medium consisted of the following components: 80 mL / L inactivated bovine serum, 800 IU / mL interleukin-5, 2 g / L Ganoderma lucidum polysaccharide, 3 g / L genistein, 40 mg / L glutathione, 80 mg / L L-galacturonic acid, 40 mg / L rhamnose galacturonic acid, 0.5 mg / L acetaminophen, 8 mg / L basic fibroblast growth factor, 8 mg / L TGF-β, 400 mg / L HEPES, 50 mL / L FBS, and the remainder being DEME medium.

[0047] (2) Preparation of biological scaffolds:

[0048] (21) Chitosan was placed in a plasma reaction vessel and treated for 4 minutes at a voltage of 220V, ambient temperature and pressure (one atmosphere) and a power of 600W. The chitosan was then removed to obtain pretreated chitosan.

[0049] Two parts of 3-glycidyl etheroxypropyltriethoxysilane and three parts of phenyltris(dimethylsiloxane)silane were added to 195 parts of acetone and mixed evenly to obtain the modified solution.

[0050] 100 parts of pretreated chitosan and 20 parts of calcium 3-hydroxy-3-methylbutyrate were added to the modification solution, stirred evenly in a 70°C water bath, ultrasonically treated at 600W for 30 minutes, filtered, and dried to obtain modified chitosan. The prepared modified chitosan is as follows: Figure 1 As shown, chitosan is a porous material with abundant and relatively regular porosity, which can provide an environment for stem cells to attach and grow.

[0051] (22) Hyaluronic acid (molecular weight 1.8 million) was added to water to prepare a hyaluronic acid solution with a mass concentration of 10%. Modified chitosan was added to water, dispersed evenly, and ultrasonically treated to obtain the precursor. The mass ratio of hyaluronic acid to modified chitosan was 1:4.

[0052] (23) Polycaprolactone was dissolved in chloroform to prepare a polycaprolactone solution with a mass concentration of 20%. The precursor was added and stirred evenly. Sodium chloride was added and stirred evenly. The solution was ultrasonically degassed and injected into a polytetrafluoroethylene mold. The solution was left to stand for 48 hours, then demolded and left to stand for another 24 hours. Finally, it was vacuum dried for 24 hours to obtain a biological scaffold.

[0053] The mass ratio of polycaprolactone, precursor, and sodium chloride is 75:25:2.

[0054] (3) The biological scaffold was treated with 75% alcohol for 2.5 hours, washed with PBS, irradiated under UV light overnight, and 1 mL of adipose stem cell suspension was evenly injected onto the biological scaffold. The scaffold was cultured in DMEM low-glucose medium for 7 days to obtain a product with biological tissue repair function.

[0055] Example 2

[0056] The application of stem cells in the preparation of a product with biological tissue repair function, wherein the product with biological tissue repair function (adipose-derived stem cells combined with a biological scaffold) includes the following steps:

[0057] (1) Preparation of adipose-derived stem cell suspension:

[0058] Adipose tissue was taken from the subcutaneous groin area of ​​SD rats, rinsed with Hanks' solution, and then minced with scissors to a volume of 1 mm. 3 Add 0.16% type II collagenase to digest for 30 min, centrifuge at 3000 rpm for 10 min, resuspend the precipitate in PBS to obtain the initial cell suspension;

[0059] The initial cell suspension was placed in culture medium and cultured for 3 days. The medium was changed to remove non-adherent cells. After culturing for another 7 days, P1 generation adipose-derived stem cells were obtained.

[0060] P1 generation adipose-derived stem cells were passaged to the 5th generation to obtain adipose-derived stem cell suspension.

[0061] The culture medium consisted of the following components: 70 mL / L inactivated bovine serum, 900 IU / mL interleukin-5, 3 g / L Ganoderma lucidum polysaccharide, 2 g / L genistein, 30 mg / L glutathione, 60 mg / L L-galacturonic acid, 45 mg / L rhamnose galacturonic acid, 0.4 mg / L acetaminophen, 6 mg / L basic fibroblast growth factor, 7 mg / L TGF-β, 200 mg / L HEPES, 40 mL / L FBS, and the remainder being DEME medium.

[0062] (2) Preparation of biological scaffolds:

[0063] (21) Chitosan was placed in a plasma reaction vessel and treated for 5 minutes at a voltage of 220V, room temperature and pressure (one atmosphere) and a power of 700W. The chitosan was then removed to obtain pretreated chitosan.

[0064] Add 3 parts of 3-glycidyl etheroxypropyltriethoxysilane and 2 parts of phenyltris(dimethylsiloxane)silane to 195 parts of acetone, mix well, and obtain the modified solution;

[0065] 100 parts of pretreated chitosan and 20 parts of calcium 3-hydroxy-3-methylbutyrate were added to the modification solution, stirred evenly in a water bath at 70°C, ultrasonically treated with 600W for 30 minutes, filtered, and dried to obtain modified chitosan.

[0066] (22) Hyaluronic acid (molecular weight 1.8 million) was added to water to prepare a hyaluronic acid solution with a mass concentration of 10%. Modified chitosan was added to water, dispersed evenly, and ultrasonically treated to obtain the precursor. The mass ratio of hyaluronic acid to modified chitosan was 1:4.

[0067] (23) Polycaprolactone was dissolved in chloroform to prepare a polycaprolactone solution with a mass concentration of 20%. The precursor was added and stirred evenly. Sodium chloride was added and stirred evenly. The solution was ultrasonically degassed and injected into a polytetrafluoroethylene mold. The solution was left to stand for 48 hours, then demolded and left to stand for another 24 hours. Finally, it was vacuum dried for 24 hours to obtain a biological scaffold.

[0068] The mass ratio of polycaprolactone, precursor, and sodium chloride is 80:20:2.

[0069] (3) The biological scaffold was treated with 75% alcohol for 2.5 hours, washed with PBS, irradiated under UV light overnight, and 1 mL of adipose stem cell suspension was evenly injected onto the biological scaffold. The scaffold was cultured in DMEM low-glucose medium for 7 days to obtain a product with biological tissue repair function.

[0070] Example 3

[0071] The application of stem cells in the preparation of a product with biological tissue repair function, wherein the product with biological tissue repair function (adipose-derived stem cells combined with a biological scaffold) includes the following steps:

[0072] (1) Preparation of adipose-derived stem cell suspension:

[0073] Adipose tissue was taken from the subcutaneous groin area of ​​SD rats, rinsed with Hanks' solution, and then minced with scissors to a volume of 1 mm. 3 Add 0.16% type II collagenase to digest for 30 min, centrifuge at 3000 rpm for 10 min, resuspend the precipitate in PBS to obtain the initial cell suspension;

[0074] The initial cell suspension was placed in culture medium and cultured for 3 days. The medium was changed to remove non-adherent cells. After culturing for another 7 days, P1 generation adipose-derived stem cells were obtained.

[0075] P1 generation adipose-derived stem cells were passaged to the 5th generation to obtain adipose-derived stem cell suspension.

[0076] The culture medium consisted of the following components: 65 mL / L inactivated bovine serum, 700 IU / mL interleukin-5, 2.5 g / L Ganoderma lucidum polysaccharide, 2.5 g / L genistein, 25 mg / L glutathione, 50 mg / L L-galacturonic acid, 35 mg / L rhamnose galacturonic acid, 0.5 mg / L acetaminophen, 5 mg / L basic fibroblast growth factor, 8 mg / L TGF-β, 300 mg / L HEPES, 45 mL / L FBS, and the remainder being DEME medium.

[0077] (2) Preparation of biological scaffolds:

[0078] (21) Chitosan was placed in a plasma reaction vessel and treated for 5 minutes at a voltage of 220V, room temperature and pressure (one atmosphere) and a power of 700W. The chitosan was then removed to obtain pretreated chitosan.

[0079] 2.5 parts of 3-glycidyl etheroxypropyltriethoxysilane and 2.5 parts of phenyltris(dimethylsiloxane)silane were added to 195 parts of acetone and mixed evenly to obtain the modified solution.

[0080] 100 parts of pretreated chitosan and 25 parts of calcium 3-hydroxy-3-methylbutyrate were added to the modification solution, stirred evenly in a water bath at 70°C, ultrasonically treated at 600W for 30 minutes, filtered, and dried to obtain modified chitosan.

[0081] (22) Hyaluronic acid (molecular weight 1.8 million) was added to water to prepare a hyaluronic acid solution with a mass concentration of 10%. Modified chitosan was added to water, dispersed evenly, and ultrasonically treated to obtain the precursor. The mass ratio of hyaluronic acid to modified chitosan was 1:3.

[0082] (23) Polycaprolactone was dissolved in chloroform to prepare a polycaprolactone solution with a mass concentration of 20%. The precursor was added and stirred evenly. Sodium chloride was added and stirred evenly. The solution was ultrasonically degassed and injected into a polytetrafluoroethylene mold. The solution was left to stand for 48 hours, then demolded and left to stand for another 24 hours. Finally, it was vacuum dried for 24 hours to obtain a biological scaffold.

[0083] The mass ratio of polycaprolactone, precursor, and sodium chloride is 70:30:2.

[0084] (3) The biological scaffold was treated with 75% alcohol for 2.5 hours, washed with PBS, irradiated under UV light overnight, and 1 mL of adipose stem cell suspension was evenly injected onto the biological scaffold. The scaffold was cultured in DMEM low-glucose medium for 7 days to obtain a product with biological tissue repair function.

[0085] Comparative Example 1

[0086] Comparative Example 1 used chitosan instead of modified chitosan, while everything else remained the same.

[0087] Comparative Example 2

[0088] The difference between Comparative Example 2 and Example 1 is that calcium 3-hydroxy-3-methylbutyrate is not added in the preparation method of the modified chitosan in Comparative Example 2.

[0089] Test case

[0090] 1. Cell proliferation detection

[0091] The number of cells proliferating was detected using CCK8 medium detection reagent.

[0092] 2. Immunofluorescence detection

[0093] Place the chitosan-polylactide scaffold on a coverslip and put it in a culture dish. Add adipose-derived stem cells at a density of 2*10 cm⁻¹. 4The cells were seeded at high density on chitosan-polylactide scaffolds and incubated with standard culture medium. After 2 days of incubation, fixation was performed with 4% paraformaldehyde for 12 min, followed by rinsing three times with PBS. Immobilization was then performed with 0.1% Triton-X100 for 2 min, followed by blocking with 1% BSA for 30 min. Primary anti-tubulin antibody was added, and the cells were incubated overnight at 4°C. After rinsing three times with PBS containing 1% serum albumin, labeled goat anti-mouse IgG secondary antibody (1:1000 1% BSA:PBS) was added, and the cells were incubated at room temperature for 45 min. After rinsing three times with PBS, DAPI (1:5000 1% BSA:PBS) was added, and nuclei were stained for 10 min at room temperature. After washing with PBS, the cells were photographed using a fluorescence microscope.

[0094] 3. Real-time reverse transcription PCR reaction

[0095] (1) RNA extraction

[0096] Total RNA was extracted from mouse skin tissue samples following the relevant steps of the Trizol assay. After extraction, the quality of the RNA was assessed using a spectrophotometer. The results showed high RNA purity and few impurities, making it suitable for the next stage of reverse transcription.

[0097] (2) Reverse transcription to synthesize cDNA

[0098] The reaction mixture consisted of: 2 μl of Oligo dT Primer (25 μM), 1 μl of dNTPMix (10 mM), and 1.5 μg of total RNA.

[0099] RNase-free dH2O up to 10 μl

[0100] 5*RT Master Mix 4μl

[0101] RNA template 2.5μg

[0102] Nuclease-free ater up to 20μl

[0103] PCR reaction procedure:

[0104] Reverse transcription to synthesize cDNA reaction program: 37℃ (15 min), 50℃ (5 min), 98℃ (5 min) 4℃ (5 min).

[0105] Real-time quantitative PCR

[0106] This experiment used SYBR Green Master mixed solution as the reaction system for real-time quantitative PCR. The reaction system was as follows:

[0107] SYBR Green Master Mixture 30μl

[0108] 0.6 μl of forward primer

[0109] 0.6 μl of reverse primer

[0110] 4 μl of cDNA sample

[0111] Nuclease-free H2O 14.8 μl

[0112] The reaction procedure for quantitative real-time PCR:

[0113] 95℃ for 10s; 93℃ (5s, 40 cycles); 65℃ (35s, 40 cycles).

[0114] Analysis of the results of quantitative real-time PCR

[0115] The relative expression level of each target gene is used as 2 -AACt Methods were calculated. The amplification efficiency of the primer pairs was first verified, and those with a difference of less than 3% from the amplification efficiency of the internal reference gene were used for quantitative comparison.

[0116] 4. Animal experiments

[0117] All laboratory animal handling followed US guidelines for laboratory animal husbandry and use. All efforts were made to minimize pain and minimize mouse use. Mice were shaved, and a 1cm x 1cm full-thickness skin incision was made on their backs. The wounds were randomly assigned to four groups (three wounds per group at each time point): 1. Control group (autologous repair group); 2. Adipose-derived stem cell group; 3. Chitosan-polylactide scaffold group; 4. Adipose-derived stem cell plus chitosan-polylactide scaffold group. All wounds were covered with a transparent material and secured with bandages. Animals were housed under identical conditions, and wound healing and animal condition were observed daily, including changes in wound area, local inflammation, granulation tissue proliferation, epithelial healing, and post-healing skin condition. On day 12, mice were euthanized using CO2 inhalation, and specimens were collected for histological and real-time quantitative PCR analysis. Skin function was assessed after wound healing.

[0118] 5. HE staining

[0119] Full-thickness skin tissue, including a 1cm radius on both sides, was harvested from the back incision site, along with normal skin tissue from the control group. The tissue was fixed with 4% paraformic acid and embedded in paraffin to prepare paraffin-embedded sections for HE staining. The specific steps are as follows:

[0120] ① Before dewaxing, place the paraffin slices in the oven for 30 minutes.

[0121] ② Dewaxing is performed by sequentially adding xylene I and xylene II for 10 minutes each.

[0122] ③Immerse the sample in 100% ethanol I, 100% ethanol II, 95% ethanol and 85% ethanol for 1 minute each to perform an alcohol gradient wash.

[0123] ④ Soak in tap water for 10 minutes.

[0124] ⑤ After staining with hematoxylin for 5 minutes, rinse with tap water for 1 minute.

[0125] ⑧ After differentiating with 1% hydrochloric acid ethanol solution for 10 seconds, rinse with tap water for 1 minute.

[0126] ⑦ After using diluted ammonia to restore the blue color for 30 seconds, rinse with tap water for 5 minutes.

[0127] ⑨ Stain with eosin solution for 3 minutes, then rinse with tap water for 1 minute.

[0128] ⑨ Dehydration using an alcohol gradient: 85% hexanol for 20 seconds, 95% alcohol for 1 minute, 100% ethanol I for 1 minute, and 100% ethanol II for 2 minutes.

[0129] Finally, xylene II for 5 minutes, then xylene II for 10 minutes. This process was then used to achieve transparency.

[0130] Finally, the slides were mounted with neutral resin, and the staining results were observed under a light microscope.

[0131] 6. Post-healing wound inspection

[0132] On day 12 post-traumatic injury, the wound area was traced using a transparent film to calculate the wound healing rate and observe the healing process. Wound healing rate (%) = (initial wound area - current wound area) / initial wound area * 100%. Immediately after trauma, the area was traced using a sterilized transparent film applied tightly to the wound edge, marking the initial wound area. The area was then scanned. Subsequent wound markings were performed using the same method. Finally, the scanned images were processed and analyzed using software; the results are shown in Table 1.

[0133] On day 12, the thickness of the healed skin in each group was measured using a microscale and observed with HE staining, as shown in Table 1.

[0134] Table 1

[0135]

[0136] As can be seen from Table 1, the scaffold described in this invention can effectively promote wound healing and increase the thickness of the healing skin. The results show that the scaffold of this invention has a good tissue repair effect.

[0137] Comparing Example 1 with Comparative Examples 1 and 2, it can be seen that modifying chitosan and adding calcium 3-hydroxy-3-methylbutyrate during the modification process further enhances the tissue repair effect.

[0138] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the technical spirit of the present invention. The technical scope of the present invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. An application of stem cells in the preparation of products with biological tissue repair functions, characterized in that, Includes the following steps: Adipose-derived stem cell suspension is injected onto a biological scaffold, cultured and proliferated to obtain a product with biological tissue repair function; The material of the biological scaffold is hyaluronic acid-modified chitosan-polycaprolactone; The method for preparing the modified chitosan includes the following steps: Chitosan was subjected to plasma treatment to obtain pretreated chitosan; 3-glycidyl etheroxypropyltriethoxysilane and phenyltris(dimethylsiloxane)silane were added to acetone and mixed evenly to obtain the modified solution. Pretreated chitosan and calcium 3-hydroxy-3-methylbutyrate were added to the modification solution, stirred evenly in a water bath at 55~80℃, ultrasonically treated, filtered, and dried to obtain modified chitosan.

2. The application according to claim 1, characterized in that, The method for preparing the adipose-derived stem cell suspension includes the following steps: Adipose tissue was taken from the subcutaneous groin area of ​​SD rats, rinsed, minced, digested, centrifuged, and resuspended in PBS to obtain a preliminary cell suspension. The cell suspension was placed in culture medium and cultured for 2-4 days. The medium was changed to remove non-adherent cells. After culturing for another 4-8 days, P1 generation adipose-derived stem cells were obtained. P1 generation adipose-derived stem cells were passaged to the 5th generation to obtain an adipose-derived stem cell suspension.

3. The application according to claim 2, characterized in that, The rinsing is performed using Hanks solution; and / or The digestion process utilizes type II collagenase.

4. The application according to claim 2, characterized in that, The culture medium comprises the following components: 60-100 mL / L inactivated bovine serum, 600-1000 IU / mL interleukin-5, 1-5 g / L Ganoderma lucidum polysaccharide, 1-5 g / L genistein, 10-100 mg / L glutathione, 20-100 mg / L L-galacturonic acid, 10-50 mg / L rhamnose galacturonic acid, 0.1-1 mg / L acetaminophen, 1-10 mg / L basic fibroblast growth factor, 1-10 mg / L TGF-β, 100-500 mg / L HEPES, 20-200 mL / L FBS, and the remainder DEME medium.

5. The application according to claim 4, characterized in that, The culture medium comprises the following components: 80 mL / L inactivated bovine serum, 800 IU / mL interleukin-5, 2 g / L Ganoderma lucidum polysaccharide, 3 g / L genistein, 40 mg / L glutathione, 80 mg / L L-galacturonic acid, 40 mg / L rhamnose galacturonic acid, 0.5 mg / L acetaminophen, 8 mg / L basic fibroblast growth factor, 8 mg / L TGF-β, 400 mg / L HEPES, 50 mL / L FBS, and the remainder being DEME medium.

6. The application according to claim 2, characterized in that, The culture was carried out at 35°C and 5% CO2 concentration.

7. The application according to claim 1, characterized in that, The method for preparing the biological scaffold includes the following steps: (1) Hyaluronic acid is added to water to prepare a hyaluronic acid solution with a mass concentration of 5-20%. Modified chitosan is added to water, dispersed evenly, and ultrasonically treated to obtain the precursor. (2) Dissolve polycaprolactone in chloroform to prepare a polycaprolactone solution with a mass concentration of 15-25%, add the precursor, stir evenly, add sodium chloride, stir evenly, degas by ultrasonication, inject into a polytetrafluoroethylene mold, let stand for 36-48 hours, then demold, let stand for another 18-36 hours, and finally vacuum dry for 24-36 hours to obtain a biological scaffold.

8. The application according to claim 7, characterized in that, In the method for preparing the biological scaffold, the mass ratio of hyaluronic acid to modified chitosan is 1:(2~5); and / or The mass ratio of polycaprolactone to the precursor is (70~85):(15~30).

9. The application according to claim 1, characterized in that, The plasma treatment power is 400~1200W, and the time is 0.5~5min; and / or The mass ratio of the pretreated chitosan, calcium 3-hydroxy-3-methylbutyrate, 3-glycidyl etheroxypropyltriethoxysilane, and phenyltris(dimethylsiloxane)silane is 1:(0.1~0.4):(0.01~0.05):(0.01~0.05).

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