A cell membrane sheet and its preparation method and application

Abstract

The present invention discloses a cell membrane sheet, its preparation method, and its application, relating to the field of biomedicine. The cell membrane sheet of the present invention comprises: a mesenchymal stem cell composite membrane sheet, including a mesenchymal stem cell membrane sheet that has been induced to undergo angiogenesis; the mesenchymal stem cell membrane sheet is pretreated with nano-reduced graphene oxide, the nano-reduced graphene oxide being prepared by reducing graphene oxide with a Rhodiola rosea extract; and an endothelial cell composite membrane sheet, including an endothelial cell membrane sheet; the endothelial cell composite membrane sheet is laminated and connected to the mesenchymal stem cell composite membrane sheet. The cell membrane sheet can promote angiogenesis and tissue repair and regeneration in tissue defect areas, and has great application prospects in the management and treatment of periodontal diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a cell membrane sheet and a preparation method and application thereof. Background Art

[0002] Periodontitis is the primary cause of periodontal bone loss, ultimately leading to tooth mobility and even tooth loss. While comprehensive periodontal treatment can improve the local inflammatory environment, it cannot restore the damaged bone tissue. In these cases, exogenous bone-stimulating constructs are needed to support gingival tissue and promote local bone regeneration.

[0003] Cell sheet technology, a key component of tissue engineering, allows for adhesion to defective tissue surfaces by preserving the endogenous cellular environment and extracellular matrix without supporting materials. Compared to two-dimensional culture conditions, three-dimensional cell sheets contain more extracellular matrix and stronger cell interactions, which can facilitate the diffusion of oxygen and nutrients through thicker tissues. Furthermore, pre-vascularized three-dimensional cell sheets can integrate with tissue vasculature early in the construct, improving the survival rate of tissue engineering grafts while also shortening defect repair time. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a cell membrane sheet that can promote angiogenesis and tissue repair and regeneration in tissue defect areas.

[0005] The present invention also provides application of nano-reduced graphene oxide prepared by reduction of Rhodiola rosea extract.

[0006] The present invention also provides a method for preparing the cell membrane sheet.

[0007] The present invention also provides applications of the cell membrane sheet.

[0008] The cell membrane sheet according to the first embodiment of the present invention comprises:

[0009] A mesenchymal stem cell composite membrane sheet, comprising a mesenchymal stem cell membrane sheet induced into angiogenesis; the mesenchymal stem cell membrane sheet is pretreated with nano-reduced graphene oxide, wherein the nano-reduced graphene oxide is prepared by reducing graphene oxide with Rhodiola rosea extract;

[0010] The endothelial cell composite membrane sheet comprises an endothelial cell membrane sheet; the endothelial cell composite membrane sheet is stacked and connected on the mesenchymal stem cell composite membrane sheet.

[0011] The cell membrane sheet according to the embodiment of the present invention has at least the following beneficial effects:

[0012] The cell membrane of the embodiment does not contain any foreign scaffold material, has good mechanical strength, is easy to operate, and has a mature and simple preparation process. It uses mesenchymal stem cells to form an early vascular network, and can use mesenchymal stem cell paracrine to promote endothelial cell angiogenesis, thereby improving the survival rate of the cell membrane after implantation and prolonging the duration of action; it can provide new blood vessels for tissue regeneration, help the formation of early vascular networks after implantation, promote the regeneration of vascularized tissues such as the oral cavity, shorten the repair time of oral tissue defects, promote the regeneration of defective bone, preserve bone tissue height, and can be applied to periodontal tissue to extend the use time of teeth, which is beneficial to improving the long-term implant effect and has good long-term biosafety. The cell membrane of the embodiment has good application prospects in the treatment and treatment of periodontal diseases.

[0013] According to some embodiments of the present invention, the thickness of the cell membrane sheet is 25 μm to 2.5 mm. Those skilled in the art can select the thickness of the cell membrane sheet as needed to match the thickness of the defect area. If the cell membrane sheet is too thin, it will not be able to support the soft tissue and will not provide sufficient space for repair. If the cell membrane sheet is too thick, it will affect the suture closure of the soft tissue.

[0014] According to some embodiments of the present invention, the mesenchymal stem cell membrane has 1 to 5 layers.

[0015] According to some embodiments of the present invention, the number of layers comprising the endothelial cell membrane sheet is 1 to 5 layers.

[0016] According to some embodiments of the present invention, the mesenchymal stem cells include at least one of deciduous tooth dental pulp stem cells, dental pulp stem cells, and bone marrow mesenchymal stem cells. Deciduous tooth dental pulp stem cells and dental pulp stem cells are less likely to cause ethical controversy, are abundant, have low immunogenicity, and have strong stemness, and can be passaged for 10 to 20 generations.

[0017] According to some embodiments of the present invention, the deciduous dental pulp stem cells are isolated from retained deciduous teeth or deciduous teeth that are decayed but not close to the pulp.

[0018] According to some embodiments of the present invention, the dental pulp stem cells are isolated from human third molars, premolars that need to be extracted for orthodontic treatment, or supernumerary teeth.

[0019] According to some embodiments of the present invention, the mesenchymal stem cells are primary cells, and the number of passages of the mesenchymal stem cells is 3 to 8.

[0020] According to some embodiments of the present invention, the endothelial cells include at least one of umbilical vein endothelial cells and microvascular endothelial cells. Compared with other endothelial cells, umbilical vein endothelial cells have higher efficacy.

[0021] According to some embodiments of the present invention, the endothelial cells are primary cells, and the number of passages of the endothelial cells is 3 to 10.

[0022] According to some embodiments of the present invention, the source of the mesenchymal stem cells or endothelial cells includes mammals, including but not limited to humans, monkeys, pigs, horses, rabbits, rats, or mice.

[0023] According to some embodiments of the present invention, the pretreatment includes: co-culturing mesenchymal stem cells and the nano-reduced graphene oxide.

[0024] According to some embodiments of the present invention, the nano-reduced graphene oxide is in the form of flakes.

[0025] According to some embodiments of the present invention, the sheet diameter of the nano-reduced graphene oxide is 40nm to 110nm. Therefore, when used to induce the differentiation of mesenchymal stem cells, there is no sudden release of degradation products caused by the degradation of the scaffold form and the toxic effects caused by long-term retention, so it has better long-term biosafety. And the nano-reduced graphene oxide within this sheet diameter range can retain physiological mechanical stimulation after contacting the cell biomembrane without causing mechanical damage to the biomembrane; and the probability of being taken up by cells is higher, and it can directly activate and regulate the signal pathways related to endothelial differentiation and cell adhesion and proliferation of deciduous dental pulp stem cells in the cells. The two-dimensional structure of the nano-reduced graphene oxide also causes it to gradually degrade in the cytoplasm after being taken up by cells, and the degradation rate is relatively stable. Moreover, since its own elemental composition is all essential elements for organisms, its biosafety is guaranteed.

[0026] According to some embodiments of the present invention, the thickness of the nano-reduced graphene oxide is 0.8 nm to 1.5 nm.

[0027] According to some embodiments of the present invention, the Rhodiola rosea extract comprises at least one of an ethanol extract of Rhodiola rosea and an aqueous extract of Rhodiola rosea. The preparation method of the Rhodiola rosea extract includes, but is not limited to, water extraction, alcohol extraction, flash extraction, enzyme extraction, supercritical extraction, or microwave-assisted extraction.

[0028] According to some embodiments of the present invention, the method for preparing the Rhodiola rosea ethanol extract comprises the following steps:

[0029] The Rhodiola rosea solution is mixed with an ethanol aqueous solution and extracted at 65-75° C. for 1.5-3 hours. The mass volume ratio of the Rhodiola rosea solution to the ethanol aqueous solution is 1 g: 15-25 mL. The volume percentage of ethanol in the ethanol aqueous solution is 75-95%.

[0030] According to some embodiments of the present invention, the method for preparing nano-reduced graphene oxide comprises the following steps:

[0031] The Rhodiola rosea extract is mixed with graphene oxide and reacted to obtain the nano-reduced graphene oxide.

[0032] Nano-reduced graphene oxide is prepared using a green reduction method. Compared to other methods, the process is safe, has no toxic byproducts, is time-efficient, and has high reduction efficiency. Nano-reduced graphene oxide not only promotes cell adhesion and proliferation, accelerating the formation of cell membranes, but also promotes the angiogenic differentiation of mesenchymal stem cells through mechanical stimulation, forming pre-vascularized mesenchymal stem cell membranes.

[0033] According to some embodiments of the present invention, the mass ratio of the Rhodiola rosea extract to graphene oxide is 1:8 to 60. For example, it can be 1:8, 1:10, 1:15, 1:20, 1:25, 1:30, 1:40, 1:50 or 1:60.

[0034] According to some embodiments of the present invention, the appropriate area of the cell membrane sheet can be selected as needed. For example, the area can be 1.5 to 2 cm 2 , 3.5~4.5cm 2 or 8.5~9.6cm 2 .

[0035] According to some embodiments of the present invention, the reaction conditions of the Rhodiola rosea extract and the graphene oxide include: reacting at 30-50° C. for 6-8 hours, and then continuing to react at 75-85° C. for 0.5-2 hours.

[0036] According to some embodiments of the present invention, the method for preparing nano-reduced graphene oxide further includes post-reaction treatment, wherein the post-reaction treatment includes at least one of solid-liquid separation, washing, and drying.

[0037] According to the second embodiment of the present invention, nano-reduced graphene oxide prepared by reducing Rhodiola rosea extract is used in the preparation of products that promote the angiogenic differentiation of deciduous dental pulp stem cells or in promoting the angiogenic differentiation of deciduous dental pulp stem cells for non-disease diagnosis and treatment purposes.

[0038] According to some embodiments of the present invention, the product is selected from a drug, a reagent or a kit.

[0039] The method for preparing the cell membrane sheet according to the third embodiment of the present invention comprises the following steps:

[0040] S1. Preparing the mesenchymal stem cell composite membrane:

[0041] Co-culturing the mesenchymal stem cells and the nano-reduced graphene oxide in an angiogenesis induction medium, and then transferring the culture to a mixed medium to obtain the mesenchymal stem cell composite membrane;

[0042] The mixed culture medium includes angiogenesis induction culture medium and membrane formation culture medium;

[0043] S2. Preparing the endothelial cell composite membrane:

[0044] culturing endothelial cells in a membrane-forming culture medium to obtain the endothelial cell composite membrane sheet;

[0045] S3. Preparing the cell membrane sheet:

[0046] The mesenchymal stem cell composite membrane sheet is brought into contact with and integrated with the endothelial cell composite membrane sheet, and cultured in angiogenesis induction medium to obtain the cell membrane sheet.

[0047] According to some embodiments of the present invention, in step S1, the concentration of the nano-reduced graphene oxide is 1 μg / mL to 5 μg / mL.

[0048] According to some embodiments of the present invention, in step S1, the number of days of co-culture is 3 to 5 days. Those skilled in the art can select the number of days of co-culture as needed based on the cell status. For example, the culture medium can be replaced with the mixed culture medium on the third day; or, alternatively, a half-volume medium change can be performed on the third day, replacing approximately half the volume of the culture medium with angiogenesis induction medium, followed by a further 2 days of culture before replacing the culture medium with the mixed culture medium.

[0049] According to some embodiments of the present invention, step S1 further comprises at least one of a pre-co-culture treatment and a post-co-culture treatment. According to some embodiments of the present invention, in the mixed culture medium, the volume ratio of the angiogenesis induction medium to the membrane formation medium is 1 to 1.5:1.

[0050] According to some embodiments of the present invention, the co-culture pre-treatment comprises: 5 ~1.5×10 5 cells / cm 2 Inoculate and culture for 12 to 24 hours.

[0051] According to some embodiments of the present invention, the co-culture post-processing includes: contacting and stacking multiple layers of mesenchymal stem cell membranes.

[0052] According to some embodiments of the present invention, the number of days of culturing in the mixed culture medium in step S1 is 2 to 5 days.

[0053] According to some embodiments of the present invention, the angiogenesis induction medium comprises serum, endothelial cell growth factor and a first basal medium.

[0054] According to some embodiments of the present invention, the angiogenesis induction medium comprises 5-12% serum, 0.8%-1.2% endothelial cell growth factor and a first culture medium.

[0055] According to some embodiments of the present invention, the first basal culture medium comprises at least one of a low-glucose DMEM medium and an α-MEM medium. The first basal culture medium refers to a culture medium that can support the growth of mesenchymal stem cells without adding special culture medium additives.

[0056] According to some embodiments of the present invention, step S2 further includes at least one of pre-culture treatment and post-culture treatment. The pre-culture treatment includes: 5 ~4.5×10 5 cells / cm 2 Inoculate and culture for 12 to 24 hours.

[0057] According to some embodiments of the present invention, in step S2, the culturing time is 3 to 5 days.

[0058] According to some embodiments of the present invention, the film-forming medium comprises serum, L-glutamine, ascorbic acid and a second basal medium.

[0059] According to some embodiments of the present invention, the film-forming culture medium comprises 5-10% FBS, 0.8-1.2% L-glutamine, 40-60 mg / L ascorbic acid and a second basal culture medium.

[0060] According to some embodiments of the present invention, the film-forming culture medium further comprises a penicillin-streptomycin dual antibody solution, wherein the amount of the penicillin-streptomycin dual antibody solution added is 0-1%.

[0061] According to some embodiments of the present invention, the second basal culture medium comprises at least one of EGM2 medium, high-glucose DMEM medium, and DMEM / F12 medium. The second basal culture medium refers to a culture medium that can support the growth of endothelial cells without adding special culture medium additives.

[0062] According to some embodiments of the present invention, the culture conditions of the cells in the preparation method independently include 36-37° C., 90%-95% humidity, and 4.8-5.2% CO 2 .

[0063] According to some embodiments of the present invention, the post-culture treatment includes: contacting and stacking multiple layers of endothelial cell membrane sheets.

[0064] According to some embodiments of the present invention, in step S3, the integration time is 24 hours to 48 hours.

[0065] According to some embodiments of the present invention, in step S3, the culture time in the angiogenesis induction medium is 5 to 7 days.

[0066] According to some embodiments of the present invention, depending on the required cell membrane strength requirements, the contact stacking method of the mesenchymal stem cell membrane or endothelial cell membrane can be selected from centrifugation (centrifugation conditions can be: 800 rpm, 37°C for 5 minutes) or manual stacking (without centrifugation).

[0067] According to the fourth aspect of the present invention, the cell membrane sheet or the preparation method is used in the preparation of bone tissue engineering repair materials or the preparation of drugs for promoting periodontal tissue regeneration.

[0068] According to some embodiments of the present invention, the drug is used to treat periodontal diseases (such as periodontitis, etc.). The cell membrane uses oral mesenchymal stem cells as the main seed cells and extracellular matrix donor, which can reduce immunogenicity and accelerate tissue regeneration.

[0069] According to some embodiments of the present invention, when the drug is used to treat patients with periodontitis (excluding contraindications of systemic diseases, completing basic periodontal treatment, and having angular bone defects in the affected teeth), the method of use is as follows:

[0070] Perform local oral anesthesia, complete oral disinfection, make an intrasulcal incision and a midline incision on the gingival papilla of the affected tooth with bone defect, open the flap, thoroughly clean the bone and tooth surfaces of the bone defect area, rinse with normal saline, and fully expose the bone and tooth surfaces of the defect area. Carefully transfer the above-mentioned cell membrane to the bone defect area, ensuring that the endothelial cell membrane part faces the bone defect area and the mesenchymal stem cell membrane part faces the soft tissue area. The cell membrane completely covers the bone defect area and leaves a 2mm edge to overlap with the healthy alveolar bone to avoid displacement of the cell membrane during soft tissue closure or oral movement, resulting in exposure of the bone defect area. The soft tissue is sutured without tension. After surgery, use antibiotics and chlorhexidine-containing mouthwash to rinse, and use systemic and oral anti-infection. The doctor advises not to squeeze the surgical area.

[0071] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0073] Figure 1 Transmission electron microscopy image of nano-reduced graphene oxide prepared by reduction of Rhodiola rosea extract;

[0074] Figure 2Atomic force microscopy morphology of nano-reduced graphene oxide prepared by reduction of Rhodiola rosea extract;

[0075] Figure 3 This is the atomic force microscopy morphology of nano-reduced graphene oxide prepared by reduction of Rhodiola rosea extract after cell uptake;

[0076] Figure 4 Schematic diagram of the preparation process of cell membrane sheets;

[0077] Figure 5 Effects of nano-reduced graphene oxide prepared by different reduction methods on endothelial differentiation of deciduous dental pulp stem cells (Sheds); ** indicates a very significant difference compared with the control group (p < 0.01), *** indicates a very significant difference compared with the control group (p < 0.001);

[0078] Figure 6 Effects of nano-reduced graphene oxide prepared by reduction of Rhodiola rosea extract on endothelial differentiation of different mesenchymal stem cells; ns indicates no significant difference compared with the control group (p>0.05), ** indicates extremely significant difference compared with the control group (p<0.01);

[0079] Figure 7 The promoting effect of the cell membrane sheets of Examples 1 to 3 and Comparative Example 1 on angiogenesis; *** indicates a very significant difference compared with the control group (p < 0.001), ## indicates a very significant difference compared with the Sheds-based group (p < 0.01), and ### indicates a very significant difference compared with the Sheds-based group (p < 0.001);

[0080] Figure 8 The repair effect of the cell membrane sheets of Examples 1 to 3 and Comparative Example 1 on bone defects; * indicates a significant difference compared with the control group (p < 0.05), ** indicates a very significant difference compared with the control group (p < 0.01), ## indicates a very significant difference compared with the Sheds-based group (p < 0.01), and ### indicates a very significant difference compared with the Sheds-based group (p < 0.001);

[0081] Figure 9 These are the H&E staining results of bone tissue of Example 1 and Comparative Example 1 after 8 weeks of implantation of the cell membrane sheets into the mandibular defect area. DETAILED DESCRIPTION

[0082] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0083] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0084] In the description of the present invention, if there is a description of first, second, etc., it is only for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0085] In the description of the present invention, it should be understood that descriptions involving orientation, such as the orientation or positional relationship indicated by up, down, etc., are based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0086] In the description of the present invention, it should be noted that, unless otherwise clearly defined, terms such as setting and connecting should be understood in a broad sense, and technicians in the relevant technical field can reasonably determine the specific meaning of the above terms in the present invention in combination with the specific content of the technical solution.

[0087] In the description of the present invention, the terms "comprises" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method or product comprising a series of steps or elements is not necessarily limited to those steps or elements explicitly listed, but may include other steps or elements not explicitly listed or inherent to such processes, methods or products.

[0088] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein are understood to include any and all subranges subsumed therein.

[0089] "And / or" is used to indicate that one or both of the stated situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0090] 1. Isolation, culture and identification of human deciduous tooth pulp stem cells (Sheds):

[0091] Freshly extracted, intact, caries-free deciduous teeth were collected and repeatedly washed with PBS containing 1% anti-D-antibody. The teeth were split open, and the pulp was removed. The apical third of the pulp was removed and washed with PBS containing 1% anti-D-antibody. The pulp tissue was minced and digested in 3 mg / mL type I collagenase for 40 min in a 37°C cell culture incubator. Digestion was terminated by adding α-MEM medium containing 10% FBS and 1% anti-D-antibody. The cells were centrifuged at 1000 rpm for 10 min, and the supernatant was discarded. The tissue pellet was evenly spread and plated in a T25 culture flask. After incubation overnight at 37°C, 95% relative humidity, and 5% CO2, the flasks were inverted and cultured as usual. After 3 days, the medium was changed halfway, and every other day. When the cells reached 80% confluency, they were digested with 0.25% trypsin and plated or subcultured. The osteogenic, chondrogenic, and adipogenic capacities of the cells were assessed, and the expression of CD29, CD44, CD105, CD90, and CD45 was determined by flow cytometry.

[0092] 2. Isolation, culture and identification of human dental pulp stem cells (DPSCs):

[0093] Freshly extracted, intact, caries-free immature permanent teeth were collected and repeatedly washed with PBS containing 1% double-antibody. The teeth were split open, and the pulp was removed. The apical third of the pulp was removed and washed with PBS containing 1% double-antibody. The pulp tissue was minced and digested in 3 mg / mL type I collagenase for 40 min in a 37°C cell culture incubator. Digestion was terminated by adding α-MEM medium containing 10% FBS and 1% double-antibody. The cells were centrifuged at 1000 rpm for 10 min, and the supernatant was discarded. The tissue pellet was evenly spread and plated in a T25 culture flask. After incubation overnight at 37°C, 95% relative humidity, and 5% CO2, the flasks were inverted and cultured as usual. After 3 days, the medium was changed halfway, and every other day. When the cells reached 80% confluency, they were digested with 0.25% trypsin and plated or subcultured. Their osteogenic, chondrogenic, and adipogenic potential was assessed, and the expression of CD29, CD44, CD105, CD90, and CD45 was assessed by flow cytometry.

[0094] 3. Isolation and culture of human umbilical vein endothelial cells:

[0095] The umbilical vein from the sterile umbilical cord was isolated and repeatedly rinsed with PBS to remove blood. The lower end of the umbilical vein was clamped with surgical forceps. 3 mg / mL collagenase was injected intravenously, and the cells were tied tightly at both ends and placed in a 37°C cell culture incubator for digestion for 30 minutes with frequent shaking. The digestion fluid was collected and rinsed with sterile PBS. The rinse fluid was collected and the cells were collected by centrifugation. After washing twice with cell culture medium, the cells were resuspended in EGM2 medium (CC-3162, Lonza Bioscience, USA) and evenly spread in a T25 culture flask. The cells were cultured at 37°C, 95% relative humidity, and 5% CO2. After one day, the culture medium and non-adherent cells were removed, the cells were rinsed with PBS, and fresh culture medium was added. The culture medium was changed every other day. When the cells reached 80% confluency, they were digested with 0.25% trypsin and then plated or subcultured.

[0096] 4. Human bone marrow mesenchymal stem cells (BMSCs) were purchased from Wuhan Punosai Life Science Technology Co., Ltd., Cat No.: CP-H166, and were identified by CD29 or CD90 immunofluorescence.

[0097] 5. Preparation of nano-reduced graphene oxide by reduction of Rhodiola rosea extract:

[0098] (1) Preparation of Rhodiola rosea extract: Rhodiola rosea was washed with high-purity water, dried at 50°C, crushed, and passed through a 60-mesh sieve; 95% ethanol (Rhodiola rosea: 95% ethanol = 1 g: 20 mL) was added, and the mixture was extracted three times in a 70°C water bath for 30 min each time. The residue was filtered and the three extracts were combined; the combined extracts were evaporated to half of their original volume using a vacuum rotary evaporator at 50°C to obtain the Rhodiola rosea extract with a final concentration of 5 g / L (based on dry matter content), which was diluted to 500 mg / L with deionized water for later use.

[0099] (2) 0.5 g of graphene oxide was added to 100 mL of 500 mg / L Rhodiola rosea extract, mixed well, and then stirred under magnetic stirring at 40°C for 8 h. After vigorous mixing, the temperature was raised to 80°C and the magnetic stirring reaction was continued for 1 h. After the reaction was completed, the mixture was centrifuged for 20 min, the precipitate was collected, washed three times with pure water, and dried in a vacuum oven at 60°C for 24 h to obtain black reduced graphene oxide.

[0100] Transmission electron microscopy and atomic force microscopy showed that the average diameter of the nano-reduced graphene oxide prepared by reduction of Rhodiola rosea extract was about 100nm, and the average thickness was about 1nm. Figure 1 and Figure 2 shown.

[0101] After treating Sheds with nano-reduced graphene oxide prepared by reduction with Rhodiola rosea extract for 24 hours, the cells were lysed, and after washing and freeze-drying, the reduced graphene oxide taken up by the cells was obtained and characterized using atomic force microscopy. Figure 3 As shown in Figure 2, after being taken up by cells, nano-reduced graphene oxide can be further degraded into smaller flakes to ensure biosafety.

[0102] 6. Preparation of nano-reduced graphene oxide by vitamin C reduction:

[0103] 0.5g of graphene oxide and 0.5g of vitamin C powder were placed in 100mL of deionized water. The mixture was ultrasonically treated at room temperature for 4 hours to achieve uniform mixing. The mixture was then heated in a 90°C water bath for 8 hours. After the reaction, the mixture was centrifuged for 20 minutes, and the precipitate was collected, washed three times with pure water, and dried in a vacuum oven at 60°C for 24 hours to obtain nano-reduced graphene oxide.

[0104] 7. Preparation of nano-reduced graphene oxide by hydrazine hydrate reduction:

[0105] 0.5 g of graphene oxide was added to 100 mL of deionized water and ultrasonicated for 1 hour to uniformly disperse it. Then, 5 mL of hydrazine hydrate was added and the mixture was ultrasonicated for 10 minutes at room temperature. The mixture was then heated in an 80°C water bath for 2 hours. After the reaction, the mixture was centrifuged for 20 minutes, and the precipitate was collected, washed three times with pure water, and dried in a vacuum oven at 60°C for 24 hours to obtain nano-reduced graphene oxide.

[0106] In the examples of the present invention, the angiogenesis induction medium used is EGM2 medium containing 10% FBS and 1% endothelial growth factor;

[0107] The film-forming medium used was EGM2 medium containing 7.5% FBS, 1% penicillin-streptomycin double antibody solution, 1% L-glutamine, and 100 mg / L ascorbic acid.

[0108] Example 1

[0109] This embodiment provides a cell membrane sheet (Sheds-based), which is composed of a deciduous tooth dental pulp stem cell composite membrane sheet (basal layer) and an umbilical vein endothelial cell composite membrane sheet (overlay layer) stacked in sequence.

[0110] The steps of preparing the cell membrane sheet are as follows:

[0111] S1. Preparation of deciduous tooth dental pulp stem cell composite membrane:

[0112] Primary human deciduous dental pulp stem cells with a confluence of 60% and passage number 3 were cultured at a rate of 2.5×10 5The cells were inoculated into 24-well culture plates at a rate of 5 cells / well, with a total of 5 wells inoculated. After culturing in α-MEM medium for 24 hours, the medium was replaced with angiogenesis induction medium containing 5 μg / mL nano-reduced graphene oxide (prepared by reduction of Rhodiola rosea extract), 500 μL / well. After 2 days (day 3), the medium was replaced with a mixed medium without nano-reduced graphene oxide (prepared by mixing angiogenesis induction medium and membrane-forming medium in a volume ratio of 1:1). The cells were cultured continuously at 37°C, 95% relative saturated humidity, and 5% CO2 for 5 days. The deciduous dental pulp stem cell sheets in the 5 wells were collected and stacked with a cell scraper to construct a deciduous dental pulp stem cell composite membrane sheet (containing 5 layers of deciduous dental pulp stem cell membrane sheets).

[0113] Nano-reduced graphene oxide was dispersed in 100 mL of deionized water to prepare a 100 μg / mL aqueous solution. Ultrasonic dispersion (40 kHz, 180 W) was then performed for 72 hours to obtain a stable aqueous solution of nano-reduced graphene oxide. This solution was then diluted with angiogenesis induction medium to obtain the appropriate concentration of the treatment reagent.

[0114] S2. Preparation of umbilical vein endothelial cell composite membrane:

[0115] Primary human umbilical vein endothelial cells with a confluence of 60% and passage number 3 were selected and cultured at a rate of 5×10 5 Cells were seeded at a density of 100 cells / well into 24-well culture plates, with a total of 2 wells inoculated. After culturing in EGM2 medium for 24 hours, the cells were replaced with membrane-forming medium and cultured for another 3 days until the umbilical vein endothelial cells fused and formed a membrane sheet. The umbilical vein endothelial cell sheets from the two wells were collected and stacked to construct a composite umbilical vein endothelial cell membrane sheet (containing two layers of umbilical vein endothelial cell sheets).

[0116] S3, prepare cell membrane sheets;

[0117] The umbilical cord vein endothelial cell composite membrane was transferred to the deciduous tooth dental pulp stem cell composite membrane. After the two composite cell membranes were in contact and integrated for 24 hours, the culture medium was replaced with angiogenesis induction medium and cultured for 5 days to obtain a cell membrane (about 1 mm thick).

[0118] Example 2

[0119] This embodiment provides a cell membrane (DPSCs-based), which is composed of a dental pulp stem cell composite membrane and an umbilical vein endothelial cell composite membrane stacked in sequence.

[0120] The preparation method of the cell membrane sheet is the same as that of Example 1, except that primary human deciduous tooth dental pulp stem cells are replaced with primary human dental pulp stem cells.

[0121] Example 3

[0122] This embodiment provides a cell membrane (BMSCs-based), which is composed of a bone marrow mesenchymal stem cell composite membrane and an umbilical vein endothelial cell composite membrane stacked in sequence.

[0123] The preparation method of the cell membrane sheet is the same as that of Example 1, except that the primary human deciduous tooth pulp stem cells are replaced with human bone marrow mesenchymal stem cells.

[0124] Comparative Example 1

[0125] This comparative example provides a cell membrane sheet, which is composed of a deciduous tooth dental pulp stem cell composite membrane sheet and an umbilical cord vein endothelial cell composite membrane sheet stacked in sequence.

[0126] The preparation method of the cell membrane sheet is the same as that of Example 1, except that in step S1, when preparing the human deciduous tooth dental pulp stem cell composite membrane sheet, nano-reduced graphene oxide is omitted.

[0127] Test Example 1

[0128] This test example tests the oxygen content of graphene oxide or nano-reduced graphene oxide prepared by different reduction methods. The test method is as follows:

[0129] The content of oxygen-containing functional groups on the sample surface was characterized using XPS equipped with a monochromatic Al Kα source (aluminum anode, 1486.68 eV, 15 kV). A 5 mg sample was placed on the sample stage for detection. After charge correction and Shirley background subtraction, CasaXPS software was used to quantitatively distinguish the different carbon components within the C1s peak according to the Gaussian-Lorentz curve: CC (284.6 eV), C-OH (285.8 eV), COC (286.6 eV), and C=O (288.2 eV).

[0130] The results are shown in Table 1.

[0131] Table 1

[0132]

[0133] Test Example 2

[0134] This test example examines the effects of nano-reduced graphene oxide prepared by different reduction methods on the endothelial differentiation of mesenchymal stem cells. The test method is as follows:

[0135] Press 2.5×10 5Shed cells / well were seeded into cell culture plates and cultured in α-MEM medium for 24 h. In experimental group 1 (hydrazine hydrate reduction), the medium was replaced with angiogenesis induction medium containing 5 μg / mL nano-reduced graphene oxide prepared by hydrazine hydrate reduction. In experimental group 2 (Vitamin C reduction), the medium was replaced with angiogenesis induction medium containing 5 μg / mL nano-reduced graphene oxide prepared by vitamin C reduction. In experimental group 3 (Rhodiola rosea reduction), the medium was replaced with angiogenesis induction medium containing 5 μg / mL nano-reduced graphene oxide prepared by Rhodiola rosea extract reduction. In the control group (CTRL), the medium was replaced with angiogenesis induction medium without nano-reduced graphene oxide. After three days of treatment, the medium was replaced with angiogenesis induction medium, and half the volume of the medium was changed every two days. On the seventh day, the cells were collected using Trizol, RNA was extracted, and the gene transcription level of CD31, an angiogenesis differentiation indicator in the cells, was detected.

[0136] The results are shown in Table 2 and Figure 5 shown.

[0137] Table 2

[0138]

[0139] The effect of nano-reduced graphene oxide prepared by reduction with Rhodiola rosea extract on increasing CD31 gene transcription levels in sheds was significantly greater than that of nano-reduced graphene oxide prepared by reduction with hydrazine hydrate or vitamin C. Among them, nano-reduced graphene oxide prepared by reduction with hydrazine hydrate did not affect the differentiation of sheds.

[0140] Test Example 3

[0141] This test example tests the effect of nano-reduced graphene oxide prepared by reducing Rhodiola rosea extract on the endothelial differentiation of different mesenchymal stem cells. The test method is as follows:

[0142] Press 2.5×10 5 DPSCs, Sheds, and BMSCs were seeded into cell culture plates at 100 cells / well. After culturing for 24 hours, the culture medium of the experimental group (rGO) was replaced with angiogenesis induction medium containing 5 μg / mL nano-reduced graphene oxide, and the culture medium of the control group (CTRL) was replaced with angiogenesis induction medium without nano-reduced graphene oxide. After three days of treatment, the culture medium was replaced with angiogenesis induction medium, and half the volume of the medium was changed every two days. On the seventh day, the cells were collected using Trizol, RNA was extracted, and the gene transcription level of CD31, an angiogenesis differentiation indicator in the cells, was detected.

[0143] The results are shown in Table 3 and Figure 6 shown.

[0144] Table 2

[0145]

[0146] Reduced graphene oxide prepared by reduction of Rhodiola rosea extract can selectively promote the transcription of CD31 gene in Sheds, while having no effect on the transcription of CD31 gene in DPSCs and BMSCs. Reduced graphene oxide prepared by reduction of Rhodiola rosea extract can selectively induce and enhance the angiogenic differentiation ability of Sheds.

[0147] Test Example 4

[0148] This test example tests the angiogenesis-promoting effect of the cell membrane sheets of Examples 1 to 3. The steps are as follows:

[0149] Twelve SD male rats weighing about 200-220 g were randomly divided into four groups and anesthetized by intraperitoneal injection. The right mandibular area was prepared and covered with drapes, and disinfected with iodine and alcohol. After local anesthesia of the surgical area, an extraoral incision of about 2 cm in length was made along the lower edge of the right mandible, and the subcutaneous fascia was separated layer by layer, the masseter fascia was cut, the periosteum was peeled off, and the bone surface was exposed. A fast turbine was used to carefully grind off the mesial and buccal alveolar bone of the mandibular first molar, and the periodontal membrane and cementum on the root surface were completely removed to establish a periodontal defect area with a volume of about 3 mm × 2 mm × 1 mm. During the operation, a large amount of normal saline was used for irrigation, and the cell membrane sheet of Example 1, the cell membrane sheet of Example 2, the cell membrane sheet of Example 3 or the cell membrane sheet of Comparative Example 1 was implanted in the defect. The bone defect area of the rats in the control group was not treated. The masseter muscle was repositioned and the sutures were sutured in layers. Antibiotics were injected continuously for 3 days after the operation to prevent infection. Two weeks after the operation, before the experimental rats were killed, the whole body was perfused with contrast agent to mark the formation of microvessels. After the experimental rats were killed, their mandibles were removed, fixed, and decalcified step by step. Micro-CT three-dimensional reconstruction was performed, and the angiogenesis was analyzed by the area of new blood vessels.

[0150] The results are as follows Figure 7 shown.

[0151] The cell sheets of Examples 1 to 3 all have angiogenesis-promoting effects and can significantly increase the area of new blood vessels. Among them, the angiogenesis-promoting effect of the cell sheet of Example 1 is significantly better than that of the cell sheets of Examples 2 to 3 and Comparative Example 1.

[0152] Test Example 5

[0153] This test example tests the bone defect repair effect of the cell membrane sheets of Examples 1 to 3 and Comparative Example 1. The steps are as follows:

[0154] Twelve SD male rats weighing about 200-220 g were randomly divided into four groups and anesthetized by intraperitoneal injection. The right mandibular area was prepared and draped, and disinfected with iodine and alcohol. After local anesthesia of the surgical area, an extraoral incision of about 2 cm in length was made along the lower edge of the right mandible, and the subcutaneous fascia was separated layer by layer, the masseter fascia was cut, the periosteum was peeled off, and the bone surface was exposed. A fast turbine was used to carefully grind off the mesial and buccal alveolar bone of the mandibular first molar, and the periodontal membrane and cementum on the root surface were completely removed to establish a periodontal defect area with a volume of about 3 mm × 2 mm × 1 mm. During the operation, a large amount of normal saline was used for flushing, and the cell membrane sheet of Example 1, the cell membrane sheet of Example 2, the cell membrane sheet of Example 3 or the cell membrane sheet of Comparative Example 1 was implanted in the defect. The bone defect area of the rats in the control group was not treated. The masseter muscle was repositioned and the sutures were sutured in layers. Antibiotics were continuously injected for 3 days after the operation to prevent infection. Eight weeks after surgery, the mandibles of the experimental rats were removed, fixed, and then decalcified in stages. Micro-CT three-dimensional reconstruction was performed, and osteogenesis was analyzed by measuring the area of new bone formation. The paraffin-embedded bone tissue was sectioned and stained with H&E, and the newly formed bone in the defect area was observed under a microscope.

[0155] The results are as follows Figure 8 and Figure 9 shown.

[0156] The cell membrane sheets of Examples 1 to 3 all had bone defect repairing effects, and the new bone area increased significantly. Among them, the bone defect repairing effect of the cell membrane sheet of Example 1 was significantly better than that of the cell membrane sheets of Examples 2 to 3 and Comparative Example 1.

[0157] The embodiments of the present invention are described in detail above in conjunction with the embodiments, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. Application of nano-reduced graphene oxide prepared by reduction of Rhodiola rosea extract in the preparation of products that promote the angiogenic differentiation of deciduous dental pulp stem cells or in promoting the angiogenic differentiation of deciduous dental pulp stem cells for non-disease diagnosis and treatment purposes.

2. A cell membrane sheet, characterized in that: include: A mesenchymal stem cell composite membrane sheet, comprising a mesenchymal stem cell membrane sheet induced to form an angiogenesis-induced mesenchymal stem cell; the mesenchymal stem cell membrane sheet is pretreated with nano-reduced graphene oxide, wherein the nano-reduced graphene oxide is prepared by reducing graphene oxide with Rhodiola rosea extract; the mesenchymal stem cells include deciduous tooth pulp stem cells; The endothelial cell composite membrane sheet comprises an endothelial cell membrane sheet; the endothelial cell composite membrane sheet is stacked and connected on the mesenchymal stem cell composite membrane sheet.

3. The cell membrane sheet according to claim 2, wherein The endothelial cells include at least one of umbilical vein endothelial cells and microvascular endothelial cells.

4. The cell membrane sheet according to claim 2, wherein The preparation method of the nano-reduced graphene oxide comprises the following steps: The Rhodiola rosea extract is mixed with graphene oxide and reacted to obtain the nano-reduced graphene oxide.

5. The cell membrane sheet according to claim 2, wherein The Rhodiola rosea extract comprises at least one of a Rhodiola rosea ethanol extract and a Rhodiola rosea water extract.

6. The cell membrane sheet according to claim 2, characterized in that The number of layers of the mesenchymal stem cell membrane is 1 to 5; And / or, the number of layers of the endothelial cell sheet is 1 to 5.

7. The method for preparing a cell membrane sheet according to any one of claims 2 to 6, characterized in that: The following steps are involved: S1. Preparing the mesenchymal stem cell composite membrane: After co-culturing the mesenchymal stem cells and the nano-reduced graphene oxide in angiogenesis induction medium, the cells are transferred to a mixed culture medium for culture to obtain the mesenchymal stem cell composite membrane; the mesenchymal stem cells include deciduous tooth pulp stem cells; The mixed culture medium includes angiogenesis induction culture medium and membrane formation culture medium; S2. Preparing the endothelial cell composite membrane: culturing endothelial cells in a membrane-forming culture medium to obtain the endothelial cell composite membrane sheet; S3. Preparing the cell membrane sheet: The mesenchymal stem cell composite membrane sheet is brought into contact with and integrated with the endothelial cell composite membrane sheet, and cultured in angiogenesis induction culture medium to obtain the cell membrane sheet.

8. The preparation method according to claim 7, characterized in that The angiogenesis induction medium comprises serum, endothelial cell growth factor and a first basal medium; And / or, the film-forming medium comprises serum, L-glutamine, ascorbic acid and a second basal medium.

9. The preparation method according to claim 7, characterized in that In step S1, the concentration of the nano-reduced graphene oxide is 1-5 μg / mL; And / or, in step S1, the co-cultivation period is 3 to 5 days.

10. The preparation method according to claim 7, characterized in that In step S3, the integration time is 24 h to 48 h; And / or, in step S3, the culture time in the angiogenesis induction medium is 5 to 7 days.

11. Use of the cell sheet according to any one of claims 2 to 6 or the cell sheet prepared by the preparation method according to any one of claims 7 to 10 in preparing bone tissue engineering repair materials or preparing drugs for promoting periodontal tissue regeneration.

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