Preparation method and application of biological agent for promoting tendon aging repair

Abstract

The invention discloses a preparation method and application of a biological agent for promoting tendon aging repair, relates to the field of treatment of tendon injury caused by aging, and aims to solve the problems of insufficient cell regeneration capacity and increase of tendon injury caused by tendon aging. The method comprises the following steps: 1, immersing activated graphene oxide aerogel into a fisetin solution, standing, and fixing to obtain fisetin-loaded graphene oxide aerogel; 2, putting the fisetin-loaded graphene oxide aerogel into a culture device, adding the single-cell suspension into the culture device, and carrying out cell culture; and 3, collecting cell supernatant, performing low-intensity pulse ultrasonic treatment, performing ultrafiltration concentration, and adding normal saline to prepare the biological preparation for promoting tendon aging repair. The biological preparation prepared by the invention can slow down tendon aging, improve tendon injury and promote tendon generation.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of tendon injury caused by aging treatment, and particularly relates to a preparation method and application of a biological agent for promoting tendon aging repair. BACKGROUND

[0002] Aging is a complex and progressive process that can lead to deterioration of tissue function, dysregulation of regenerative capacity, and ultimately impairment of body system function. Oxidative stress is an important factor leading to cell and organism aging, and during the aging process, a large number of cells and molecules change, and then the normal tissue function pathologically degenerates. In the tendon, aging leads to significant changes in the structure and composition of the extracellular matrix. In addition, the cellular environment of the aging tendon also changes, including a significant decrease in cell density and metabolic activity, and an increase in the degree of cell aging. These degenerative changes, combined, make aging a key risk factor for the development of tendonopathy and can increase the frequency of tendon injury, so aging is a key factor for tendon disease.

[0003] Tendon is composed of different cell populations, including tendon stem cells (TSCs), tenocytes, tenoblasts and vascular cells. TSCs are the precursors of tenocytes and tenoblasts and reside within a unique tendon niche. TSCs have stem cell properties, but in addition to expressing stem cell characteristic cell surface markers (Sca-1 and CD44), TSCs also highly express tendon-specific markers such as type I collagen (Col1a1), tendon transcription factor (Scx), tenomodulin (Tnmd) and tenascin C (Tnc). The regenerative capacity of tendon depends on the state of TSCs, which can activate and expand to form new tendon collagen fibers and self-renew to cope with tendon tissue damage. The TSCs stemness and regenerative capacity are significantly damaged in the tendon tissue of the elderly, leading to a decrease in the efficiency of tendon self-repair, so reasonable inhibition of the aging of TSCs is the key to slowing down the aging of tendon and repairing tendon injury. SUMMARY

[0004] The present application is to solve the problem of insufficient cell regeneration caused by tendon aging, increase tendon injury, and provide a preparation method and application of a biological agent for promoting tendon aging repair.

[0005] The preparation method of the biological agent for promoting tendon aging repair of the present application comprises the following steps:

[0006] Step one: activate the graphene oxide aerogel, immerse the activated graphene oxide aerogel in a fisetin solution, stand, fix, and obtain the graphene oxide aerogel loaded with fisetin (Fisetin-GOA);

[0007] Step two: umbilical cord mesenchymal stem cells are made into a single cell suspension;

[0008] After sterilization, the graphene oxide aerogel loaded with ombuine is placed in a cell culture device, and then the single cell suspension is added to the cell culture device containing the aerogel, and cultured in a 37℃, 5% CO2 cell incubator. After the cells adhere or attach to the aerogel, fresh medium is replaced, and fresh medium is replaced every 1d for culture;

[0009] Step three: after three days of culture, the cell supernatant obtained by culture is treated with low-intensity pulsed ultrasound, and the treatment conditions are: pulse length 200 μs, pulse repetition frequency 1 kHz, total exposure time 20-30 min, ultrafiltration concentration, to obtain a cell culture supernatant concentrate rich in tendon aging repair factors, add physiological saline, and prepare a biological preparation for promoting tendon aging repair.

[0010] Further, the standing in step one is at 4℃ for 12-15 h.

[0011] Further, the specific method of activation in step one is to soak the graphene oxide aerogel in PBS buffer for 1-2 h.

[0012] Further, the ombuine solution in step one is to dissolve ombuine in dimethyl sulfoxide (DMSO) with a concentration of 1-30 µM.

[0013] Further, the fixation method in step one is to soak the graphene oxide aerogel in 1% glutaraldehyde for 1 h.

[0014] Further, the medium for the single cell suspension in step two is MEM basic medium + 10% KSR serum substitute + 1% penicillin streptomycin.

[0015] Further, the volume ratio of the single cell suspension to the graphene oxide aerogel loaded with ombuine in step two is 3:2.

[0016] Further, the medium replaced in step two is: MEM basic medium + 10% KSR serum substitute + 1% penicillin streptomycin.

[0017] Further, the specific method of ultrafiltration concentration in step three is to transfer the cell supernatant treated with low-intensity pulsed ultrasound to an ultrafiltration tube and centrifuge at 4000xg for 10-12 min.

[0018] Further, the volume ratio of the concentrate to physiological saline in step three is 1: (10-20).

[0019] The application also provides application of the biological preparation prepared by the method in preparation of drugs for slowing down tendon aging, improving tendon injury and promoting tendon generation.

[0020] Advantages of the application:

[0021] The application utilizes the quercetin-loaded graphene oxide aerogel system to culture UMSCs, promotes the UMSCs to release a large amount of functional cytokines, and then utilizes low-intensity pulse ultrasound treatment to further activate, to obtain a biological preparation for promoting tendon aging repair. The application verifies the effect of the tendon aging repair biological preparation by combining in-vitro and in-vivo experiments. In the in-vitro experiment, adriamycin (DOX) is used to treat tendon stem cells (TSCs), to construct a tendon stem cell aging model. Subsequently, by detecting various functional indexes of the TSCs treated by the tendon aging repair factor, the in-vitro effect verification is completed. In the in-vivo experiment, collagenase is used to treat rat tendons, to establish a rat tendon aging injury model. Subsequently, by detecting the oxidative stress level and tendon function recovery of the model rats treated by the tendon aging repair biological preparation, the in-vivo effect verification is realized. The results show that the biological preparation prepared by the application can act on tendon stem cells and play the oxidative stress protection and tendon regeneration functions, slow down tendon aging and repair injury, and provide a new idea for tendon aging and injury treatment. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The percentage of SA-β-gal positive cells in TSCs (per 500 cells);

[0023] Figure 2 CCK-8 cell viability detection of TSCs;

[0024] Figure 3 P21 immunofluorescence staining result of TSCs;

[0025] Figure 4 The corresponding column chart of the fluorescence staining result; Figure 3

[0026] The mRNA expression level of the oxidative stress related gene Nrf2 of TSCs; Figure 5

[0027] The mRNA expression level of the oxidative stress related gene HO-1 of TSCs; Figure 6

[0028] The mRNA expression level of the oxidative stress related gene SOD of TSCs; Figure 7

[0029] Figure 8 ​mRNA expression level of a specific tendon-related gene Scx in the TSCs;

[0030] Figure 9 mRNA expression level of a specific tendon-related gene Tnc in the TSCs;

[0031] Figure 10 mRNA expression level of a specific tendon-related gene Tnmd in the TSCs;

[0032] Figure 11 mRNA expression level of a specific tendon-related gene Colla1 in the TSCs;

[0033] Figure 12 mRNA expression level of a tendon tissue oxidative stress-related gene Nrf2;

[0034] Figure 13 mRNA expression level of a tendon tissue oxidative stress-related gene HO-1;

[0035] Figure 14 mRNA expression level of a tendon tissue oxidative stress-related gene SOD;

[0036] Figure 15 mRNA expression level of a tendon-related gene Scx in the tendon tissue;

[0037] Figure 16 mRNA expression level of a tendon-related gene Tnc in the tendon tissue;

[0038] Figure 17 mRNA expression level of a tendon-related gene Tnmd in the tendon tissue;

[0039] Figure 18 mRNA expression level of a tendon-related gene Colla1 in the tendon tissue;

[0040] Figure 19 extreme load detection of the tendon tissue;

[0041] Figure 20 stress detection of the tendon tissue;

[0042] Figure 21 rigidity detection of the tendon tissue. DETAILED DESCRIPTION

[0043] The technical solution of the present application is not limited to the following specific embodiments, but also includes any combination of the specific embodiments.

[0044] Specific embodiment one: the preparation method of the biological agent for promoting tendon aging repair in the present embodiment includes the following steps:

[0045] Step one: activate the graphene oxide aerogel, immerse the activated graphene oxide aerogel in fisetin solution, stand at 4℃ for 12-15 h, fix, and obtain the graphene oxide aerogel loaded with fisetin (Fisetin-GOA);

[0046] Step two: prepare the umbilical cord mesenchymal stem cells into a single cell suspension of 3×10 4

[0047] After sterilizing the graphene oxide aerogel loaded with fisetin, place it in a cell culture device, then add the single cell suspension into the cell culture device containing the aerogel, and culture in a 37℃, 5% CO2 cell incubator, after the cells adhere or attach to the aerogel, replace the fresh culture medium, and replace the fresh culture medium every 1 d for culture;

[0048] Step three: after three days of culture, collect the cell supernatant obtained by culture, treat the cell supernatant by low-intensity pulsed ultrasound, the treatment conditions are: pulse length is 200 μs, pulse repetition frequency is 1 kHz, total exposure time is 20-30 min, ultrafiltration concentration, obtain the cell culture supernatant concentrate rich in tendon aging repair factors, add physiological saline, and prepare a biological preparation for promoting tendon aging repair.

[0049] In the process of UMSCs culture, the method adds fisetin to treat the cells, which can promote the cells to secrete rich tendon aging repair factors with anti-aging function, and has a good protective effect on tendon stem cells, by slowing down the aging of tendon stem cells, and thus improves tendon aging and damage.

[0050] In the method, fisetin is loaded on graphene oxide aerogel for UMSCs culture, because the graphene oxide aerogel can make fisetin continuously release, achieve a sustained effect, and thus promote the cells to secrete more tendon aging repair factors; and the graphene oxide aerogel can inhibit the occurrence of oxidative stress of cells, activate the release of antioxidant stress-related factors in cells, up-regulate the Nrf2 / HO-1 signal pathway of tendon stem cells to inhibit oxidative stress, and achieve the effect of improving tendon aging, thereby slowing down tendon aging.

[0051] In the method, the cell culture supernatant is treated by low-intensity pulsed ultrasound, which can further activate the cells, promote the specific release of cell factors that slow down tendon cell aging and damage, and thus achieve a higher effect of improving tendon aging and damage.

[0052] ​Specific implementation method two: the specific method of activation in step one of this embodiment is to soak the graphene oxide aerogel in PBS buffer for 1-2h. The other steps and parameters are the same as those in specific implementation method one.

[0053] Specific implementation method three: the solution of fisetin in step one of this embodiment is to dissolve fisetin in dimethyl sulfoxide (DMSO) with a concentration of 1-30µM. The other steps and parameters are the same as those in specific implementation method one or two.

[0054] Specific implementation method four: the fixation method in step one of this embodiment is to soak the graphene oxide aerogel in 1% glutaraldehyde for 1h. The other steps and parameters are the same as those in one of specific implementation methods one to three.

[0055] Specific implementation method five: the medium for single cell suspension in step two of this embodiment is MEM basic medium + 10% KSR serum substitute + 1% penicillin streptomycin. The other steps and parameters are the same as those in one of specific implementation methods one to four.

[0056] Specific implementation method six: the volume ratio of single cell suspension to fisetin-loaded graphene oxide aerogel in step two of this embodiment is 3:2. The other steps and parameters are the same as those in one of specific implementation methods one to five.

[0057] Specific implementation method seven: the medium for replacement in step two of this embodiment is: MEM basic medium + 10% KSR serum substitute + 1% penicillin streptomycin. The other steps and parameters are the same as those in one of specific implementation methods one to six.

[0058] Specific implementation method eight: the specific method of ultrafiltration concentration in step three of this embodiment is to transfer the cell supernatant after low-intensity pulsed ultrasound treatment to an ultrafiltration tube and centrifuge at 4000xg for 10-12min. The other steps and parameters are the same as those in one of specific implementation methods one to seven.

[0059] Specific implementation method nine: the volume ratio of concentrated solution to normal saline in step three of this embodiment is 1: (10-20). The other steps and parameters are the same as those in one of specific implementation methods one to eight.

[0060] Specific implementation method ten: the application of the biological preparation of this embodiment in the preparation of drugs for slowing down tendon aging, improving tendon injury and promoting tendon generation.

[0061] The following embodiments of the present application are described in detail. The following embodiments are implemented on the premise of the technical solutions of the present application, and detailed implementation schemes and specific operation processes are given. However, the protection scope of the present application is not limited to the following embodiments.

[0062] Example 1:

[0063] The present embodiment promotes the preparation method of biological agents for tendon aging repair, comprising the following steps:

[0064] I. Preparation of graphene oxide aerogel (GOA)

[0065] (1) Preparation of graphene oxide (GO) dispersion: graphene oxide powder was added to deionized water at a concentration of 3 mg / mL, and a uniform and stable GO dispersion was obtained by ultrasonic treatment, the power of the ultrasonic treatment was 1000 W, and the time was 60 min. Then filter with a 0.22 μm microporous filter membrane to remove large size aggregates that are not completely dispersed, to obtain a more uniform solution, and then adjust the pH value to 8 (weak alkaline) to promote the hydrophilic interaction of GO layers, which is the GO dispersion.

[0066] (2) Hydrothermal reaction: the GO dispersion was transferred to a hydrothermal reactor, and after sealing, it was reacted at 180°C for 12 h. During the reaction, the GO layers gradually self-assembled into a three-dimensional network structure of hydrogel due to the hydrogen bonding of oxygen-containing functional groups (hydroxyl, carboxyl) and the π-π stacking effect.

[0067] (3) Drying treatment: freeze-drying the hydrogel at -80°C for 24 h, and then sublimating the water in a vacuum environment to obtain graphene oxide aerogel (GOA) with high porosity.

[0068] II. Preparation method of GOA loaded with fisetin (Fisetin-GOA)

[0069] The graphene oxide aerogel was soaked in PBS buffer for 1 h to activate the surface functional groups, and then the activated graphene oxide aerogel was immersed in a 10 µM fisetin solution (fisetin dissolved in DMSO) at 4°C for 12 h, and then fixed with 1% glutaraldehyde for 1 h to obtain graphene oxide aerogel loaded with fisetin (Fisetin-GOA). Fisetin-GOA can be stored at -80°C.

[0070] III. Obtaining of cell culture supernatant

[0071] The Wharton's jelly was separated from the umbilical cord tissue, and UMSCs were obtained by the method of cell scraping. P5 generation of UMSCs in good growth state were taken for cell counting, and a single cell suspension (3×10 4 The culture medium used was culture medium A: MEM basic culture medium + 10% KSR serum substitute + 1% penicillin streptomycin.

[0072] Fisetin-GOA was made into a cylinder with a diameter of 5 mm and a height of 5 mm, and after high-temperature and high-pressure sterilization, it was placed in a sterile 96-well plate for standby.

[0073] Take 150 μL of single cell suspension and place it into the small hole containing the Fisetin-GOA cylinder. Place the 96-well plate in a 37°C, 5% CO2 cell incubator for 24 hours. After the cells adhere or attach to the Fisetin-GOA cylinder, replace the fresh culture medium A. Replace the fresh culture medium A every 1 day thereafter for 3 days.

[0074] The culture medium A is: MEM basic medium + 10% KSR serum substitute + 1% penicillin streptomycin.

[0075] Four, preparation of tendon aging repair biological agent

[0076] Collect the MSCs supernatant obtained by culture in step three, and treat the supernatant with low-intensity pulsed ultrasound (LIPUS) under the following conditions: pulse length of 200 μs, pulse repetition frequency of 1 kHz, and total exposure time of 30 min. Then transfer to a 50 mL 100KD ultrafiltration tube, centrifuge at 4000 x g for 10 min for ultrafiltration concentration, obtain the cell culture supernatant concentrate rich in tendon aging repair factors, add physiological saline, and prepare into a tendon aging repair biological agent (AF), wherein the volume ratio of the concentrate to physiological saline is 1:10.

[0077] To verify the beneficial effects of the UMSCs culture method in step three of this embodiment, a comparative experiment was conducted as follows:

[0078] Comparative Example A: P5 generation UMSCs were cultured with culture medium A, i.e. single cell suspension (3 x 10 4 was placed in a sterile 96-well plate and placed in a 37°C, 5% CO2 cell incubator for 24 hours. After the cells adhered, fresh culture medium A was replaced, and fresh culture medium A was replaced every 1 day thereafter for 3 days.

[0079] The culture medium A is: MEM basic medium + 10% KSR serum substitute + 1% penicillin streptomycin.

[0080] Comparative Example B: P5 generation UMSCs were cultured with culture medium B, i.e. single cell suspension (3 x 10 4 was placed in a sterile 96-well plate and placed in a 37°C, 5% CO2 cell incubator for 24 hours. After the cells adhered, fresh culture medium B was replaced, and fresh culture medium B was replaced every 1 day thereafter for 3 days.

[0081] The culture medium B is: MEM basic medium + 10 μM Fisetin + 10% KSR serum replacement + 1% penicillin streptomycin.

[0082] Comparative Example C: P5 generation UMSCs were cultured by using culture medium A, and graphene oxide aerogel (GOA) was made into a cylinder with a diameter of 5 mm and a height of 5 mm, and after high-temperature and high-pressure sterilization, it was placed in a sterile 96-well plate for standby.

[0083] Take 150 μL single cell suspension into the small hole containing GOA, and place the 96-well plate in a 37℃, 5% CO2 cell incubator for 24h, and after the cells adhere or adhere to the GOA, replace the fresh culture medium A, and replace the fresh culture medium A every 1d thereafter, and culture for 3 days.

[0084] The culture medium A is: MEM basic medium + 10% KSR serum replacement + 1% penicillin streptomycin.

[0085] UMSCs can secrete a variety of bioactive molecules, including growth factors that play an important role in tendon repair: insulin-like growth factor 1 (IGF-1), platelet-derived growth factor (PDGF) and fibroblast growth factor (bFGF) and the like. These growth factors promote tendon repair by participating in intercellular information transmission. The cytokine content of the cell culture fluid obtained by the above four methods was detected by ELISA, and the cytokine secretion is shown in Table 1. Compared with the comparative example, the content of each cytokine obtained by the method of Example 1 is higher, indicating that UMSCs cultured by Fisetin-GOA system can secrete more tendon repair related cytokines.

[0086] Table 1 Change in content of tendon repair related cytokines secreted by UMSCs after culture

[0087]

[0088] Example 2:

[0089] The preparation method of the cytokine preparation of the present embodiment comprises the following steps:

[0090] I. Preparation of graphene oxide aerogel (GOA)

[0091] (1) Preparation of graphene oxide (GO) dispersion: Graphene oxide powder was added to deionized water at a concentration of 3 mg / mL, and a uniform and stable GO dispersion was obtained by ultrasonic treatment. The ultrasonic treatment power was 1000 W and the time was 60 min. Then, the solution was filtered through a 0.22 μm microporous membrane to remove large-sized agglomerates that were not completely dispersed, resulting in a more uniform solution. The pH value was then adjusted to 8 (weakly alkaline) to promote the hydrophilic interaction of the GO sheets.

[0092] (2) Hydrothermal reaction: The GO dispersion was transferred to a high-pressure reactor, sealed, and reacted at 200℃ for 12 h. During the reaction, the GO sheets gradually self-assembled into a three-dimensional network structure hydrogel due to the hydrogen bonding of oxygen-containing functional groups (hydroxyl and carboxyl groups) and the π-π stacking effect.

[0093] (3) Drying treatment: The hydrogel was frozen at -80℃ for 24 h, and then the water was removed by sublimation under vacuum to obtain graphene oxide aerogel with high porosity.

[0094] II. Preparation method of Fisetin-GOA (Fisetin-GOA)

[0095] Graphene oxide aerogel was immersed in PBS buffer for 1 h to activate its surface functional groups. The activated graphene oxide aerogel was then immersed in a 10 µM flavin solution and allowed to stand at 4 °C for 12 h. It was then fixed by immersion in 1% glutaraldehyde for 1 h to obtain flavin-loaded graphene oxide aerogel (Fisetin-GOA). Fisetin-GOA can be frozen at -80 °C.

[0096] III. Obtaining Cell Culture Supernatant

[0097] Wharton's jelly was isolated from umbilical cord tissue, and UMSCs were obtained by the smear method. Well-grown P5 generation UMSCs were collected, cell counted, and single-cell suspensions (3 × 10⁻⁶) were prepared. 4 (pcs / mL) for later use.

[0098] Fisetin-GOA was made into cylinders with a diameter of 5 mm and a height of 5 mm, and after being sterilized by high temperature and high pressure, it was placed in a sterile 96-well plate for later use.

[0099] Take 150 μL of single-cell suspension and place it into a well containing Fisetin-GOA cylinders. Place the 96-well plate in a 37℃, 5% CO2 cell culture incubator for 24 h. After the cells adhere to the wall or attach to the Fisetin-GOA cylinders, replace the medium with fresh medium. Thereafter, replace the medium with fresh medium every 1 day for further culture.

[0100] The culture medium is: MEM basic medium + 10% KSR serum substitute + 1% penicillin streptomycin.

[0101] IV. Preparation of the cytokine preparation

[0102] The MSCs supernatant obtained in step three of the culture is transferred into a 50 mL 100KD ultrafiltration tube, centrifuged at 4000xg for 10 min for ultrafiltration concentration, to obtain a cell culture supernatant concentrate, and physiological saline is added to prepare a cytokine preparation (CF), wherein the volume ratio of the concentrate to physiological saline is 1:10.

[0103] (I) Detection of the content of tendon aging repair related factors

[0104] The two preparations prepared in Example 1 and Example 2 are subjected to cytokine content detection by ELISA, and the detection results are shown in Table 2. Comparison shows that the cytokine content in the preparation after low-intensity pulsed ultrasound treatment in Example 1 is higher, indicating that low-intensity pulsed ultrasound treatment can further activate UMSCs to promote the specific release of tendon aging repair related factors.

[0105] Table 2 Change in the content of tendon aging repair related cytokines in the preparation after LIPUS treatment

[0106]

[0107] (II) Repair effect of senescent tendon stem cells

[0108] Tendon stem cells (TSCs) are isolated from rat patellar tendons, and the specific operation is as follows: the tendon is dissected, quickly immersed in 70% ethanol solution for disinfection and to kill surface cells, and the tendon tissue is cut into small pieces and placed in low-sugar DMEM containing 2 mg / mL collagenase type I and 1 mg / mL collagenase type IV for digestion for 2 hours. Then the cell suspension is filtered with a 70 μm filter and inoculated in a T-25 culture flask containing standard culture medium (low-sugar DMEM + 10% fetal bovine serum + 1% penicillin streptomycin), and the culture medium is replaced every other day, and the cells are expanded to P1 generation for subsequent experiments.

[0109] The cultured TSCs are divided into the following 4 groups:

[0110] Group a: conventional culture group (Control). TSCs are cultured with standard culture medium as a control. The standard culture medium formula is: low-sugar DMEM + 10% fetal bovine serum + 1% penicillin streptomycin.

[0111] Group b: aging model group (DOX). First, TSCs were induced to be senescent by 200 nM doxorubicin (DOX) for 72 h, and then the DOX-treated TSCs were cultured in standard medium as model control. The standard medium formula: low glucose DMEM + 10% fetal bovine serum + 1% penicillin-streptomycin.

[0112] Group c: cytokine treatment group (DOX + CF). First, TSCs were induced to be senescent by 200 nM doxorubicin (DOX) for 72 h, and then the DOX-treated TSCs were cultured in CF medium. The CF medium is prepared by adding the cytokine preparation (CF) prepared in Example 2 to the standard medium. The CF medium formula: low glucose DMEM + 5% CF (volume) + 5% fetal bovine serum + 1% penicillin-streptomycin.

[0113] Group d: tendon aging repair biological preparation treatment group (DOX + AF). First, TSCs were induced to be senescent by 200 nM doxorubicin (DOX) for 72 h, and then the DOX-treated TSCs were cultured in AF medium. The AF medium is prepared by adding the tendon aging repair biological preparation (AF) prepared in Example 1 to the standard medium. The AF medium formula: low glucose DMEM + 5% AF (volume) + 5% fetal bovine serum + 1% penicillin-streptomycin.

[0114] Each group of TSCs was cultured for 7 days, and the senescence of TSCs was detected by β-galactosidase kit and immunofluorescence. The protective effect of tendon aging repair factor on oxidative stress of TSCs (Nrf2, HO-1, SOD) and the expression of main tendon-related markers (Scx, Tnc, Tnmd, Collal) was analyzed by quantitative real-time PCR. The effect of tendon aging repair factor on tendon stem cells was evaluated.

[0115] Oxidative stress is the main cause of cell and tissue aging. In tendon tissue, oxidative stress can reduce the ability of TSCs to self-renew, proliferate and differentiate into tendon cells, and further exacerbate tendon aging. β-galactosidase (SA-β-gal) is a widely used marker for cell senescence. Because the lysosomal content in senescent cells usually increases, the activity of lysosomal enzyme β-galactosidase increases. Cell cycle regulatory protein P21, as one of the markers of cell senescence, plays a core role in the initiation and maintenance of cell senescence. The expression level of P21 can reflect the degree of cell senescence, and inhibiting the expression of P21 can slow down cell aging. The level of SA-β-Gal in cells was detected by kit, the cell viability was detected by CCK-8, and the expression of P21 in TSCs was detected by immunofluorescence. The degree of cell senescence and cell viability were evaluated. The percentage of SA-β-gal positive cells in TSCs (per 500 cells) was as follows: Figure 1As shown, the results show that the percentage of SA-β-gal positive cells in the DOX+AF group is significantly down-regulated compared with the DOX group and the DOX+C group. The CCK-8 cell viability detection results of TSCs are as shown in Figure 2 As shown, the cell viability of the DOX+AF group is shown to be increased compared with the DOX group, and is not different from the control group cells. Figure 3 and Figure 4 For the expression of P21 in TSCs, it can be seen that the positive expression of P21 is significantly increased after DOX treatment, and the positive expression of P21 is significantly decreased after AF treatment, tending to the control group of healthy cells. The above results show that the tendon aging repair biological preparation prepared by the method of the application can inhibit the cell aging caused by DOX to TSCs, and improve the cell viability.

[0116] Nuclear factor (Nrf2) is a key transcription factor for cells to cope with oxidative stress, and heme oxygenase 1 (HO-1) is a key antioxidant enzyme, which plays a key role in preventing cell and tissue damage caused by oxidative stress, and can induce the transcription of various antioxidant genes, thereby enhancing the antioxidant stress resistance of cells and tissues. Studies have shown that oxidative stress can accelerate tendon aging, resulting in a significant decrease in the expression levels of Nrf2 / HO-1 and downstream antioxidant enzymes (superoxide dismutase SOD), and up-regulation of Nrf2 / HO-1 can inhibit TSCs oxidative stress and thus slow down tendon tissue aging. The mRNA expression results of Nrf2, HO-1 and SOD in TSCs are as shown in Figures 5-7 As shown, the results show that the expression levels of Nrf2, HO-1 and SOD in the DOX group cells are significantly decreased compared with the control group TSCs after DOX treatment, and the expression levels of the three oxidative stress-related genes in the DOX+AF group are significantly increased, indicating that the tendon aging repair biological preparation can inhibit the oxidative stress of senescent TSCs.

[0117] Tnc is a glycoprotein of the extracellular matrix, which is the basis for cell-cell and cell-matrix interactions. Collagen is the main protein in tendons and provides a stretch-maintaining environment for tendon cells, and oxidative stress can reduce the expression of collagen in tissues. Tnmd is essential for early tendon differentiation, and Scx can positively regulate the expression of Tnmd and Colla1. Studies have shown that oxidative stress can impair the tendon differentiation ability of TSCs and inhibit the expression of tendon-related genes. The mRNA expression levels of Scx, Tnc, Tnmd and Colla1 in TSCs are as shown in Figures 8-11As shown, the results show that the expression levels of Scx, Tnc, Tnmd and Collal in the DOX group after DOX treatment are significantly reduced compared with the control group, while the expression levels in the DOX+AF group are significantly increased, indicating that the tendon aging repair biological preparation of the application can significantly improve the tendon differentiation function of TSCs cells after DOX treatment, protect TSCs and slow down the damage caused by aging.

[0118] (III) Treatment effect of rat tendon aging model

[0119] The experimental rats were divided into the following 4 groups:

[0120] The first group was a healthy control group (Control), and the animals were not treated.

[0121] The second group was a model control group (Model), and the collagenase was used to establish a tendon injury animal model, and then physiological saline was used for sham treatment. The treatment method was to inject 50 µL of physiological saline into the tendon injury site of the rat.

[0122] The method for establishing a tendon injury animal model was to perfuse 50 µL (700 UI) of type I collagenase into the hind limbs of 8-week-old rats, and the rat tendon injury model was established after 2 weeks.

[0123] The third group was a CF preparation treatment group (CF), and the collagenase was used to establish a tendon injury animal model, and then the CF preparation prepared in Example 2 was used for treatment. The treatment method was to inject 50 µL of CF preparation into the tendon injury site of the rat.

[0124] The fourth group was an AF preparation treatment group (AF), and the collagenase was used to establish a tendon injury animal model, and then the AF preparation prepared in Example 1 was used for treatment. The treatment method was to inject 50 µL of AF preparation into the tendon injury site of the rat.

[0125] The injection dose and time of the three groups of animals were uniform, and blood collection and tendon tissue sampling were performed after 4 weeks.

[0126] The protective effect of tendon aging repair factors on oxidative stress of rat tendons (Nrf2, HO-1, SOD) and the expression of main tendon-related markers (Scx, Tnc, Tnmd, Collal) was detected by quantitative real-time PCR analysis. The changes in ultimate load, stress and stiffness of rat tendons were evaluated by biomechanics. The improvement effect of tendon aging repair factors on aging rat tendons was evaluated.

[0127] As shown in FIG. 6, the results show that the expression levels of Scx, Tnc, Tnmd and Collal in the DOX group after DOX treatment are significantly reduced compared with the control group, while the expression levels in the DOX+AF group are significantly increased, indicating that the tendon aging repair biological preparation of the application can significantly improve the tendon differentiation function of TSCs cells after DOX treatment, protect TSCs and slow down the damage caused by aging. Figures 12-14As shown in FIG. 6, the mRNA expression levels of the oxidative stress-related genes Nrf2, HO-1 and SOD, the expression levels of Nrf2, HO-1 and SOD in the tendon tissues of the tendon injury rats of the model group were significantly decreased compared with the control group, while the expression levels of Nrf2, HO-1 and SOD in the tendon tissues of the tendon aging repair biological preparation treatment group were significantly up-regulated, indicating that the tendon aging repair biological preparation can improve the oxidative stress of the tendon tissues caused by aging. Figures 15-18 As shown in FIG. 7, the mRNA expression levels of the tendon generation-related genes Scx, Tnc, Tnmd and Colla1 in the tendon tissues, the results showed that compared with the control group, the expression levels of Scx, Tnc, Tnmd and Colla1 in the tendon tissues of the tendon injury rats of the model group were significantly decreased, while the expression levels of Scx, Tnc, Tnmd and Colla1 in the tendon aging repair biological preparation treatment group were significantly increased and significantly higher than those of the CF preparation treatment group.

[0128] One of the significant features of tendon aging is the gradual and irreversible change in its mechanical properties. This is mainly attributed to potential changes in tendon cell number, cell function, collagen turnover rate, collagen fiber diameter and arrangement, and collagen cross-linking. Generally, several parameters such as ultimate load, stress and stiffness are used to evaluate the biomechanical properties of tendons. The results of the biomechanical detection of the tendon tissues are shown in FIG. 8. Figures 19-21 As shown in FIG. 8, compared with the control group, the ultimate load, stress and stiffness of the tendon tissues of the model group were significantly decreased, while after treatment with the tendon aging repair biological preparation, the three indicators were significantly up-regulated and basically restored to be the same as the healthy control group, indicating that the tendon aging repair biological preparation can improve tendon injury and promote tendon generation.

[0129] In summary, the results show that the tendon aging repair biological preparation of the present application can adjust oxidative stress to protect tendon stem cells from injury and inhibit cell aging; by repairing the function of tendon stem cells, promote the expression of tendon generation-related genes, improve damaged cells and tendon tissues, and thus slow down tendon aging.

Claims

1. A method of preparing a biological agent to promote tendon aging repair, characterized in that, The method comprises the following steps: Step one: activating the graphene oxide aerogel, immersing the activated graphene oxide aerogel in a quercetin solution, standing, fixing, and obtaining the graphene oxide aerogel loaded with quercetin; Step two: preparing a single-cell suspension of umbilical cord mesenchymal stem cells; After sterilizing the graphene oxide aerogel loaded with quercetin, placing it in a cell culture device, then adding the single-cell suspension into the cell culture device containing the aerogel, and culturing the cells, after the cells adhere to or attach to the aerogel, replacing the fresh culture medium, and then replacing the fresh culture medium every 1 day for culture; Step three: after three days of culture, collecting the cell supernatant obtained by culture, treating the cell supernatant by low-intensity pulsed ultrasound, the treatment conditions being: a pulse length of 200 μs, a pulse repetition frequency of 1 kHz, a total exposure time of 20-30 min, ultrafiltration concentration, obtaining a cell culture supernatant concentrate rich in tendon aging repair factors, adding physiological saline, and preparing a biological preparation for promoting tendon aging repair.

2. The method for preparing a biological agent for promoting tendon aging repair according to claim 1, characterized in that, The specific method of activation in step one is: immersing the graphene oxide aerogel in PBS buffer for 1-2 h.

3. A method for preparing a biological agent for promoting tendon aging repair according to claim 1 or 2, characterized in that, The quercetin solution in step one is: dissolving quercetin in dimethyl sulfoxide, and the concentration is 1-30 µM.

4. The method for preparing a biological agent for promoting tendon aging repair according to claim 3, characterized in that, The fixing method in step one is: immersing the graphene oxide aerogel in 1% glutaraldehyde for 1 h.

5. The method for preparing a biological agent for promoting tendon aging repair according to claim 1, characterized in that, The culture medium for the single-cell suspension in step two is: MEM basic medium + 10% KSR serum substitute + 1% penicillin streptomycin.

6. The method for preparing a biological agent for promoting tendon aging repair according to claim 1, characterized in that, The volume ratio of the single-cell suspension to the graphene oxide aerogel loaded with quercetin in step two is 3:

2.

7. The method for preparing a biological agent for promoting tendon aging repair according to claim 1, characterized in that, The replaced culture medium in step two is: MEM basic medium + 10% KSR serum substitute + 1% penicillin streptomycin.

8. The method for preparing a biological agent for promoting tendon aging repair according to claim 1, characterized in that, The specific method of ultrafiltration concentration in step three is: transferring the cell supernatant treated by low-intensity pulsed ultrasound into an ultrafiltration tube, and centrifuging at 4000×g for 10-12 min.

9. A method for preparing a biological agent for promoting tendon aging repair according to claim 1, characterized in that, The volume ratio of the concentrate to physiological saline in step three is 1: (10-20).

10. The use of the biological preparation prepared by the method of claim 1 in the preparation of a drug for slowing down tendon aging, improving tendon injury, and promoting tendon generation.

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