CN118320172A - Cell patch and preparation method and application thereof - Google Patents
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a cell membrane and a preparation method and application thereof, and relates to the technical field of biological medicines. The cell patch of the present invention comprises: a mesenchymal stem cell composite membrane comprising a angioblast-induced mesenchymal stem cell membrane; the mesenchymal stem cell membrane is pretreated by nano-reduced graphene oxide, and the nano-reduced graphene oxide is prepared by reducing graphene oxide by rhodiola rosea extract; an endothelial cell composite membrane comprising an endothelial cell membrane; the endothelial cell composite membrane sheet is connected to the mesenchymal stem cell composite membrane sheet in a lamination manner. It can promote angiogenesis in tissue defect area and repair and regeneration of tissue, and has good application prospect in the aspects of treatment and treatment of periodontal diseases.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to a cell membrane and a preparation method and application thereof.
Background
Periodontitis is a major cause of periodontal bone defects, ultimately leading to loosening of teeth, and even loss of teeth. Although perfect periodontal treatment can improve the local inflammatory environment, defective bone tissue cannot be restored. At this time, it is necessary to use exogenous constructs that promote osteogenesis, support gingival tissue, and promote local bone regeneration.
Tissue engineering in which cell patch technology can adhere to the surface of defective tissue by preserving the endogenous cellular environment, extracellular matrix, without supporting materials. And compared with two-dimensional culture conditions, the three-dimensional cell patch contains more extracellular matrix and stronger cell interaction, and can promote diffusion barrier of oxygen and nutrients in thicker tissues; meanwhile, the three-dimensional cell membrane of the preformed vessel can be integrated with the tissue vessel as early as possible in the construct, so that the survival rate of the tissue engineering graft can be improved, and the defect repair time can be shortened.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, the invention provides a cell patch which can promote angiogenesis and tissue repair regeneration in a tissue defect area.
The invention also provides application of the nano reduced graphene oxide prepared by reduction of the rhodiola rosea extract.
The invention also provides a preparation method of the cell membrane.
The invention also provides application of the cell membrane.
According to an embodiment of the first aspect of the invention, a cell patch comprises:
A mesenchymal stem cell composite membrane comprising a angioblast-induced mesenchymal stem cell membrane; the mesenchymal stem cell membrane is pretreated by nano-reduced graphene oxide, and the nano-reduced graphene oxide is prepared by reducing graphene oxide by rhodiola rosea extract;
an endothelial cell composite membrane comprising an endothelial cell membrane; the endothelial cell composite membrane sheet is connected to the mesenchymal stem cell composite membrane sheet in a lamination manner.
The cell membrane according to the embodiment of the invention has at least the following beneficial effects:
The cell membrane of the embodiment has no external stent material, has better mechanical strength, is beneficial to operation, and has mature and simple preparation process. The mesenchymal stem cells are utilized to form early vascular networks, and the paracrine of the mesenchymal stem cells can be utilized to promote the vascularization of endothelial cells, so that the survival rate of the cell patch after implantation is improved and the action time is prolonged; can provide new blood vessels for tissue regeneration, is favorable for the formation of early-stage implantation blood vessel networks, promotes the regeneration of vascularized tissues such as oral cavity and the like, shortens the repair time of oral cavity tissue defects and the like, promotes the regeneration of defective bones, saves the bone tissue height, can be applied to periodontal tissues to prolong the service time of teeth, is favorable for improving long-term implantation effects, and has good long-term biological safety. The cell patch of the embodiment has good application prospect in the aspects of treatment and treatment of periodontal diseases.
According to some embodiments of the invention, the cell patch has a thickness of 25 μm to 2.5mm. The thickness of the cell patch can be selected by one skilled in the art as desired to match the cell patch thickness to the defect area thickness. If the cell membrane is too thin, the soft tissue cannot be supported, and enough repairing space is difficult to provide; if the cell patch is too thick, it can affect soft tissue suture closure.
According to some embodiments of the invention, the mesenchymal stem cell membrane has a number of layers of 1-5.
According to some embodiments of the invention, the number of layers comprising the endothelial cell membrane is 1-5.
According to some embodiments of the invention, the mesenchymal stem cells comprise at least one of deciduous dental pulp stem cells, and bone marrow mesenchymal stem cells. The deciduous teeth dental pulp stem cells and dental pulp stem cells are not easy to cause ethical disputes, are rich in sources, low in immunogenicity and strong in dryness, and can be passaged for 10-20 generations.
According to some embodiments of the invention, the deciduous dental pulp stem cells are isolated from retained deciduous teeth or decayed but not near-medullary deciduous teeth.
According to some embodiments of the invention, the dental pulp stem cells are isolated from human third molar, premolars or multi-dentures for orthodontic treatment requiring extraction.
According to some embodiments of the invention, the mesenchymal stem cells are primary cells. The passage number of the mesenchymal stem cells is 3-8.
According to some embodiments of the invention, the endothelial cells include at least one of umbilical vein endothelial cells, microvascular endothelial cells. Umbilical vein endothelial cells are more potent than other endothelial cells.
According to some embodiments of the invention, the endothelial cells are primary cells. The passage number of the endothelial cells is 3-10 generations.
According to some embodiments of the invention, the source of mesenchymal stem cells or endothelial cells comprises a mammal. The mammal includes, but is not limited to, a human, monkey, pig, horse, rabbit, rat, or mouse.
According to some embodiments of the invention, the preprocessing comprises: and co-culturing the mesenchymal stem cells with the nano-reduced graphene oxide.
According to some embodiments of the invention, the nano-reduced graphene oxide is in the form of platelets.
According to some embodiments of the invention, the sheet diameter of the nano-reduced graphene oxide is 40 nm-110 nm. Therefore, when the preparation method is used for inducing the differentiation of the mesenchymal stem cells, toxic effects caused by the sudden release and long-term retention of degradation products caused by the degradation of the scaffold form do not exist, so that the preparation method has better long-term biosafety. The nano-reduced graphene oxide within the sheet diameter range can retain physiological mechanical stimulation after contacting a cell biomembrane, and mechanical damage to the biomembrane can not be generated; the probability of being taken up by the cells is higher, and the signal channels related to differentiation, cell adhesion and proliferation of the tooth pulp stem cell endotheliosis of the deciduous teeth can be directly activated and regulated in the cells. The two-dimensional structure of the nano-reduced graphene oxide also leads to gradual degradation in cytoplasm after being taken up by cells, the degradation speed is stable, and the biological safety is ensured because the element composition of the nano-reduced graphene oxide is all necessary elements for organisms.
According to some embodiments of the invention, the thickness of the nano-reduced graphene oxide is 0.8nm to 1.5nm.
According to some embodiments of the invention, the rhodiola rosea extract comprises at least one of rhodiola rosea alcohol extract and rhodiola rosea water extract. The preparation method of the rhodiola rosea extract comprises, but is not limited to, water extraction, alcohol extraction, flash extraction, enzyme extraction, supercritical extraction or microwave-assisted extraction.
According to some embodiments of the invention, the method for preparing the rhodiola rosea alcohol extract comprises the following steps:
Mixing the rhodiola rosea with ethanol water solution, and extracting for 1.5-3 h at 65-75 ℃. The mass volume ratio of the rhodiola root to the ethanol water solution is 1g:15 mL-25 mL. The volume percentage of the ethanol in the ethanol water solution is 75-95%.
According to some embodiments of the present invention, the method for preparing nano-reduced graphene oxide includes the steps of:
Mixing the rhodiola rosea extract with graphene oxide, and reacting to obtain the nano-reduced graphene oxide.
Compared with other methods, the nano-reduction graphene oxide is prepared by a green reduction method, and has the advantages of safe process, no toxic byproducts, short time and high reduction efficiency. The nanometer reduced graphene oxide can promote cell adhesion and proliferation and accelerate cell membrane generation; meanwhile, the mesenchymal stem cell membrane can promote the angiogenic differentiation of the mesenchymal stem cells through mechanical stimulation to form the mesenchymal stem cell membrane of the preformed blood vessel.
According to some embodiments of the invention, the mass ratio of the rhodiola rosea extract to the graphene oxide is 1:8 to 60. For example: can be 1: 8. 1: 10. 1: 15. 1: 20. 1: 25. 1: 30. 1: 40. 1:50 or 1:60.
According to some embodiments of the invention, the appropriate area of the cell patch may be selected as desired. For example: the area may be 1.5 to 2cm 2,3.5～4.5cm2 or 8.5 to 9.6cm 2.
According to some embodiments of the invention, the reaction conditions of the rhodiola rosea extract and the graphene oxide comprise: after reacting for 6-8 h at 30-50 ℃, the reaction is continued for 0.5-2 h at 75-85 ℃.
According to some embodiments of the invention, the method for preparing nano-reduced graphene oxide further comprises post-reaction treatment. The post-reaction treatment comprises at least one of solid-liquid separation, cleaning and drying.
The application of the nano reduced graphene oxide prepared by the rhodiola rosea extract reduction in the preparation of a product for promoting the vascularization differentiation of the dental pulp stem cells of the deciduous teeth or for the purpose of non-disease diagnosis and treatment is provided.
According to some embodiments of the invention, the product is selected from a drug, a reagent or a kit.
According to a third aspect of the present invention, the method for preparing a cell membrane as described above includes the steps of:
S1, preparing the mesenchymal stem cell composite membrane:
Co-culturing the mesenchymal stem cells and the nano reduced graphene oxide in a angiogenesis induction culture medium, and transferring the culture medium to a mixed culture medium for culture to obtain the mesenchymal stem cell composite membrane;
the mixed culture medium comprises a angiopoiesis induction culture medium and a film forming culture medium;
s2, preparing the endothelial cell composite membrane sheet:
culturing endothelial cells in a film forming culture medium to obtain the endothelial cell composite membrane;
S3, preparing the cell membrane:
And (3) contacting and integrating the mesenchymal stem cell composite membrane with the endothelial cell composite membrane, and culturing in a vascularization induction culture medium to obtain the cell membrane.
According to some embodiments of the invention, in step S1, the concentration of the nano-reduced graphene oxide is 1 μg/mL to 5 μg/mL.
According to some embodiments of the invention, in step S1, the number of days of co-cultivation is 3 to 5 days. The number of days for co-culture can be selected by one skilled in the art as desired depending on the cell state. For example: the medium may be replaced with the mixed medium on the third day; the medium may be replaced with the mixed medium after half the volume of the medium is replaced with the vascular induction medium on the third day and the culture is continued for 2 days.
According to some embodiments of the invention, step S1 further comprises at least one of a co-cultivation pre-treatment and a co-cultivation post-treatment. According to some embodiments of the invention, in the mixed medium, the volume ratio of the vascularization induction medium to the film forming medium is 1 to 1.5:1.
According to some embodiments of the invention, the co-cultivation pre-treatment comprises: the mesenchymal stem cells are inoculated and cultured for 12 to 24 hours according to 1.1X10 5～1.5×105cells/cm2.
According to some embodiments of the invention, the co-cultivation post-treatment comprises: the multiple layers of mesenchymal stem cell membranes are subjected to contact superposition.
According to some embodiments of the invention, the number of days of culture in the mixed medium in step S1 is 2 to 5 days.
According to some embodiments of the invention, the angiogenesis-inducing medium comprises serum, endothelial cell growth factor, and a first basal medium.
According to some embodiments of the invention, the angiopoiesis induction medium comprises 5-12% serum, 0.8% -1.2% endothelial cell growth factor, and a first medium.
According to some embodiments of the invention, the first basal medium comprises at least one of a low sugar DMEM medium, an alpha-MEM medium. The first basal medium refers to a medium that can support the growth of mesenchymal stem cells without adding special medium additives.
According to some embodiments of the invention, step S2 further comprises at least one of a pre-culture treatment and a post-culture treatment. The pretreatment for culture comprises: the endothelial cells were seeded at 2.5X10 5～4.5×105cells/cm2 and cultured for 12-24 h.
According to some embodiments of the invention, in step S2, the incubation time is 3 to 5 days.
According to some embodiments of the invention, the film-forming medium comprises serum, L-glutamine, ascorbic acid, and a second basal medium.
According to some embodiments of the invention, the film-forming medium comprises 5-10% FBS, 0.8-1.2% L-glutamine, 40-60 mg/L ascorbic acid and a second basal medium.
According to some embodiments of the invention, the film-forming medium further comprises a penicillin-streptomycin diabody solution. The addition amount of the penicillin-streptomycin double antibody solution is 0-1%.
According to some embodiments of the invention, the second basal medium comprises at least one of EGM2 medium, high-sugar DMEM medium, DMEM/F12 medium. The second basal medium refers to a medium that can support the growth of endothelial cells without the addition of special medium additives.
According to some embodiments of the invention, the culture conditions of the cells in the preparation method comprise 36-37 ℃ and 90-95% humidity and 4.8-5.2% CO 2 independently.
According to some embodiments of the invention, the post-culture treatment comprises: multiple endothelial cell patches were contact stacked.
According to some embodiments of the invention, in step S3, the integration time is 24h to 48h.
According to some embodiments of the invention, in step S3, the culturing in the angiogenetic induction medium is for a period of 5 to 7 days.
According to some embodiments of the invention, the contact stacking of the mesenchymal stem cell membrane or endothelial cell membrane may be performed by centrifugation (centrifugation conditions may be 800rpm, centrifugation at 37℃for 5 min) or manual stacking (without centrifugation), depending on the desired cell membrane strength requirements.
Use of the cell membrane or the method of preparation according to the embodiment of the fourth aspect of the present invention for the preparation of a bone tissue engineering repair material or for the preparation of a medicament for promoting periodontal tissue regeneration.
According to some embodiments of the invention, the medicament is for treating periodontal disease (e.g., periodontitis, etc.). The cell patch selects the oral mesenchymal stem cells as main seed cells and extracellular matrix donors, so that the immunogenicity can be reduced, and the tissue regeneration can be accelerated.
According to some embodiments of the invention, the medicament is used for treating patients with periodontitis (excluding systemic disease contraindications, perfecting periodontal basic treatment, suffering from teeth with angular bone defects), and is used as follows:
The local anesthesia of the oral cavity is carried out, the disinfection of the oral cavity is perfected, the incision in the bone defect area bone surface and tooth surface are fully cleaned by turning over the valve, and the bone surface and tooth surface of the defect area are completely exposed by normal saline. The cell membrane is carefully transferred to the bone defect area, so that the endothelial cell membrane part faces the bone defect area, the mesenchymal stem cell membrane part faces the soft tissue area, the cell membrane completely covers the bone defect area, the edge of 2mm is reserved to be overlapped with healthy alveolar bone, and the cell membrane is prevented from being shifted in the soft tissue closing or oral cavity movement process, so that the bone defect area is exposed. Soft tissue is sutured without tension. After operation, antibiotics and chlorhexidine-containing mouthwash are used for gargling, so that the whole body and the local part of the oral cavity are anti-infective, and doctor orders not to squeeze the operation area.
Additional features and advantages of the 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 invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a transmission electron microscope image of nano reduced graphene oxide prepared by reduction of rhodiola rosea extract;
FIG. 2 is an atomic force microscopic morphology diagram of nano-reduced graphene oxide prepared by reduction of rhodiola rosea extract;
FIG. 3 is an atomic force microscopic morphology diagram of nano reduced graphene oxide prepared by reduction of rhodiola rosea extract after cell ingestion;
FIG. 4 is a schematic diagram of a preparation flow of cell membranes;
FIG. 5 is a graph showing the effect of nano-reduced graphene oxide prepared by different reduction methods on endothelial differentiation of deciduous tooth pulp stem cells (Sheds); * Indicating a significant difference (p < 0.01) from the control group, indicating a significant difference (p < 0.001) from the control group;
FIG. 6 is a graph showing the effect of nano-reduced graphene oxide prepared by reduction of rhodiola rosea extract on endothelial differentiation of different mesenchymal stem cells; ns represents no significant difference (p > 0.05) from the control group, x represents very significant difference (p < 0.01) from the control group;
FIG. 7 shows the effect of cell membranes of examples 1 to 3 and comparative example 1 on promoting angiogenesis; * Indicates a very significant difference (p < 0.001) from the control group, # indicates a very significant difference (p < 0.01) from the Sheds-based group, and # # indicates a very significant difference (p < 0.001) from the Sheds-based group;
FIG. 8 shows the repairing effect of the cell membrane of examples 1 to 3 and comparative example 1 on bone defects; * Indicating significant differences from the control group (p < 0.05), indicating significant differences from the control group (p < 0.01), # indicating significant differences from the Sheds-based group (p < 0.01), and # # indicating significant differences from the Sheds-based group (p < 0.001);
FIG. 9 shows the results of H & E staining of bone tissue 8 weeks after the cell patch of example 1 and comparative example 1 was implanted in the mandibular defect area.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In the description of the present invention, the description of first, second, etc. is for the purpose of distinguishing between technical features only and should not be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated or implicitly indicating the precedence of the technical features indicated.
In the description of the present invention, it should be understood that the direction or positional relationship indicated with respect to the description of the orientation, such as up, down, etc., is based on the direction or positional relationship shown in the drawings, is merely for convenience of describing the present invention and simplifying the description, and does not indicate or imply that the apparatus or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus should not be construed as limiting the present invention.
In the description of the present invention, unless explicitly defined otherwise, terms such as arrangement, connection, etc. should be construed broadly, and a person skilled in the art may reasonably determine the specific meaning of the terms in the present invention by combining the specific contents of the technical solutions.
In the description of the present invention, the terms "comprises" and "comprising," along with any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, or article of manufacture that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, or article of manufacture.
When a range of values is disclosed herein, the range is considered to be continuous and includes both the minimum and maximum values for the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range description features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to include any and all subranges subsumed therein.
"And/or" is used to indicate that one or both of the illustrated cases may occur, e.g., a and/or B include (a and B) and (a or B).
1. Isolation, culture and identification of human deciduous dental pulp stem cells (Sheds):
Collecting freshly extracted complete caries-free deciduous teeth, repeatedly cleaning with PBS containing 1% double antibodies, cleaving teeth, taking out dental pulp, removing 1/3 dental pulp of root tip, and cleaning with PBS containing 1% double antibodies; cutting dental pulp tissue, placing into 3mg/mL type I collagenase, digesting for 40min at 37 ℃ in a cell culture box, adding an alpha-MEM culture medium containing 10% FBS and 1% double antibody, stopping digestion, centrifuging at 1000rpm for 10min, discarding supernatant, placing tissue block precipitate into a T25 culture flask, uniformly dispersing and spreading, culturing at 37 ℃ under the condition of 95% relative saturated humidity and 5% CO 2 for one night, turning over the flask, culturing conventionally, changing liquid half after 3 days, changing liquid every 2 days, and when the cells are fused to 80%, using 0.25% pancreatin for digestion, and performing cell inoculation or subculture. The osteogenic, chondrogenic and adipogenic capacity of the cells were tested, and the CD29, CD44, CD105, CD90 and CD45 expression of the cells was tested by using flow cytometry.
2. Isolation, culture and characterization of human Dental Pulp Stem Cells (DPSCs):
Collecting freshly extracted complete caries-free young permanent teeth, repeatedly cleaning with PBS containing 1% double antibodies, cleaving teeth, taking out dental pulp, removing 1/3 dental pulp of root tip, and cleaning with PBS containing 1% double antibodies; cutting dental pulp tissue, placing into 3mg/mL type I collagenase, digesting for 40min at 37 ℃ in a cell culture box, adding an alpha-MEM culture medium containing 10% FBS and 1% double antibody, stopping digestion, centrifuging at 1000rpm for 10min, discarding supernatant, placing tissue block precipitate into a T25 culture flask, uniformly dispersing and spreading, culturing at 37 ℃ under the condition of 95% relative saturated humidity and 5% CO 2 for one night, turning over the flask, culturing conventionally, changing liquid half after 3 days, changing liquid every 2 days, and when the cells are fused to 80%, using 0.25% pancreatin for digestion, and performing cell inoculation or subculture. The osteogenic, chondrogenic and adipogenic capacity of the strain is detected, and the expression of CD29, CD44, CD105, CD90 and CD45 of the strain is detected by using flow cytometry and identified.
3. Isolation and culture of human umbilical vein endothelial cells:
separating umbilical vein in sterile umbilical cord, repeatedly washing with PBS, removing blood, clamping lower end of umbilical vein with forceps, injecting collagenase 3mg/mL into vein, fastening two ends, and placing in cell incubator at 37deg.C for digestion for 30min, and shaking up and down for several times. Collecting digestive juice, washing with sterile PBS, collecting washing liquid, centrifuging to collect cells, washing with cell culture medium for 2 times, re-suspending cells with EGM2 culture medium (CC-3162,Lonza Bioscience, USA), placing in T25 culture flask, culturing at 37deg.C, 95% relative saturated humidity, 5% CO 2, removing culture medium and non-adherent cells after 1 day, washing with PBS, adding fresh culture medium, changing liquid every other day, and digesting with 0.25% pancreatin when cells are fused to 80%, and performing cell inoculation or subculture.
4. Human bone marrow mesenchymal stem cells (BMSCs) were purchased from the Withanbozier life technologies Co., ltd, cat No. CP-H166, and identified by manufacturer's CD29 or CD90 immunofluorescence.
5. Preparing nano-reduction graphene oxide by reduction of rhodiola rosea extract:
(1) Preparing rhodiola rosea extract: cleaning radix Rhodiolae with high purity water, oven drying at 50deg.C, pulverizing, and sieving with 60 mesh sieve; adding 95% ethanol (radix Rhodiolae: 95% ethanol=1g:20ml), extracting in water bath at 70deg.C for 3 times each for 30min, filtering to remove residues, and mixing the 3 times of extractive solutions; evaporating the combined extract to half of original volume at 50deg.C by vacuum rotary evaporator to obtain radix Rhodiolae extract with final concentration of 5g/L (dry matter content), and diluting with deionized water to 500 mg/L.
(2) Adding 0.5g of graphene oxide into 100mL of rhodiola rosea extract with the concentration of 500mg/L, uniformly mixing, and then magnetically stirring at 40 ℃ for reaction for 8 hours; after being mixed vigorously, the temperature is raised to 80 ℃ and the magnetic stirring reaction is continued for 1h. And after the reaction is finished, centrifuging for 20min, collecting the precipitate, cleaning the precipitate with pure water for 3 times, and drying the precipitate in a vacuum oven at 60 ℃ for 24h to obtain black reduced graphene oxide.
The nanometer reduction graphene oxide prepared by reduction of the rhodiola rosea extract has the average diameter of about 100nm and the average thickness of about 1nm, which are characterized by a transmission electron microscope and an atomic force microscope. The results are shown in fig. 1 and 2.
Treating Sheds hours of nano reduced graphene oxide prepared by reduction of rhodiola rosea extract, then cracking cells, washing, freeze-drying and the like to obtain reduced graphene oxide taken up by the cells, and carrying out characterization by using an atomic force microscope. The results are shown in FIG. 3. After being taken up by cells, the nano-reduced graphene oxide can be further degraded into smaller pieces so as to ensure biological safety.
6. Preparing nano-reduced graphene oxide by reducing vitamin C:
0.5g of graphene oxide and 0.5g of vitamin C powder are placed into 100mL of deionized water, and the mixed solution is subjected to ultrasonic treatment for 4 hours at room temperature, so that the mixed solution is uniformly mixed. Then heating in water bath at 90 ℃ for 8 hours. And after the reaction is finished, centrifuging for 20min, collecting the precipitate, cleaning the precipitate with pure water for 3 times, and drying the precipitate in a vacuum oven at 60 ℃ for 24h to obtain the nano-reduced graphene oxide.
7. Preparing nano-reduction graphene oxide by hydrazine hydrate reduction:
0.5g of graphene oxide is added into 100mL of deionized water, after being subjected to ultrasonic treatment for 1h to be uniformly dispersed, 5mL of hydrazine hydrate is added, and the mixed solution is subjected to ultrasonic treatment at room temperature for 10min. Then heated in a water bath at 80℃for 2h. And after the reaction is finished, centrifuging for 20min, collecting the precipitate, cleaning the precipitate with pure water for 3 times, and drying the precipitate in a vacuum oven at 60 ℃ for 24h to obtain the nano-reduced graphene oxide.
In the embodiment of the invention, the angiopoiesis induction culture medium is EGM2 culture medium containing 10% FBS and 1% endothelial cell growth factor;
The film-forming medium used was EGM2 medium containing 7.5% FBS, 1% penicillin-streptomycin diabody solution, 1% L-glutamine, 100mg/L ascorbic acid.
Example 1
The present example provides a cell patch (Sheds-based) consisting of an deciduous dental pulp stem cell composite patch (basal layer) and an umbilical vein endothelial cell composite patch (superimposed layer) laminated in this order.
The preparation method of the cell patch comprises the following steps:
S1, preparing a composite membrane of dental pulp stem cells of the deciduous teeth:
inoculating primary humanized deciduous tooth pulp stem cells with fusion degree reaching 60% and passage number of 3 into 24-hole culture plate according to 2.5X10 5/hole, inoculating 5 holes altogether, culturing in alpha-MEM culture medium for 24 hr, replacing culture medium with angiogenetic induction culture medium containing 5 μg/mL nanometer reduced graphene oxide (prepared by reduction of radix Rhodiolae extract), 500 μl/hole, changing the culture medium into a mixed culture medium (obtained by mixing a vascularization induction culture medium and a film forming culture medium according to a volume ratio of 1:1) without nano-reduction graphene every 2 days (day 3), continuously culturing for 5 days under the conditions of 37 ℃ and 95% relative saturation humidity and 5% CO 2, collecting and superposing the deciduous tooth pulp stem cell membranes in 5 holes by using a cell scraper, and constructing the deciduous tooth pulp stem cell composite membrane (containing 5 layers of deciduous tooth pulp stem cell membranes).
The nano-reduced graphene oxide is dispersed in 100mL of deionized water to prepare a nano-reduced graphene oxide aqueous solution with the concentration of 100 mug/mL, and the nano-reduced graphene oxide aqueous solution is dispersed for 72 hours by ultrasonic waves (40 Khz, 180W) to obtain a stable nano-reduced graphene oxide aqueous solution. And diluting with a angiogenesis induction culture medium to obtain a treatment reagent with a corresponding concentration.
S2, preparing an umbilical vein endothelial cell composite membrane sheet:
Primary humanized umbilical vein endothelial cells with the fusion degree reaching 60% and the passage number of 3 are selected, inoculated into a 24-hole culture plate according to the density of 5 multiplied by 10 5/hole, inoculated into 2 holes altogether, cultured in an EGM2 culture medium for 24 hours, and then replaced by a film-forming culture medium to be continuously cultured for 3 days until the umbilical vein endothelial cells are fused to form a film. Umbilical vein endothelial cell patches in the 2 holes are collected and overlapped to construct an umbilical vein endothelial cell composite patch (containing 2 layers of umbilical vein endothelial cell patches).
S3, preparing cell membranes;
transferring umbilical vein endothelial cell composite membrane onto dental pulp stem cell composite membrane of deciduous teeth, contacting and integrating the two composite cell membranes for 24 hr, and changing culture solution into angiopoiesis induction culture medium, and continuously culturing for 5 days to obtain cell membrane (thickness about 1 mm).
Example 2
The present example provides a cell patch (DPSCs-based) consisting of a dental pulp stem cell composite patch and an umbilical vein endothelial cell composite patch laminated in this order.
The preparation method of the cell membrane is the same as in example 1, and only differs in that: the primary human deciduous dental pulp stem cells are replaced with primary human dental pulp stem cells.
Example 3
The present example provides a cell patch (BMSCs-based) consisting of a bone marrow mesenchymal stem cell composite patch and an umbilical vein endothelial cell composite patch laminated in this order.
The preparation method of the cell membrane is the same as in example 1, and only differs in that: replacing the primary human deciduous tooth dental pulp stem cells with human bone marrow mesenchymal stem cells.
Comparative example 1
The comparative example provides a cell patch consisting of an deciduous tooth dental pulp stem cell composite patch and an umbilical vein endothelial cell composite patch laminated in sequence.
The preparation method of the cell membrane is the same as in example 1, and only differs in that: in step S1, when the humanized deciduous tooth dental pulp stem cell composite membrane is prepared, the nano-reduced graphene oxide is defaulted.
Detection example 1
The detection example detects the oxygen content of graphene oxide or nano-reduced graphene oxide prepared by different reduction methods. The detection method comprises the following steps:
XPS, equipped with a monochromatic Al K alpha source (aluminum anode, 1486.68eV,15 kV), was used to characterize the content of oxygen-containing functional groups on the sample surface. 5mg of the sample was placed on a sample stage for detection. After charge correction, the Shirley background of the sample was subtracted and the different carbon components within the C1s peak were quantitatively distinguished according to gaussian-lorentz curve using casxps software: C-C (284.6 eV), C-OH (285.8 eV), C-O-C (286.6 eV), C=O (288.2 eV).
The results are shown in Table 1.
TABLE 1
Detection example 2
The detection example detects the influence of nano-reduced graphene oxide prepared by different reduction methods on the endothelial differentiation of mesenchymal stem cells. The detection method comprises the following steps:
Inoculating Shed into a cell culture plate according to 2.5X10 5 cells/hole, culturing for 24 hours in alpha-MEM culture medium, replacing the culture medium with a vascularization induction culture medium containing nano-reduced graphene oxide prepared by reducing hydrazine hydrate at 5 mug/mL in experiment group 1 (hydrazine hydrate reduction), replacing the culture medium with a vascularization induction culture medium containing nano-reduced graphene oxide prepared by reducing vitamin C at 5 mug/mL in experiment group 2 (Vitamin C reduction), replacing the culture medium with a vascularization induction culture medium containing nano-reduced graphene oxide prepared by reducing rhodiola rosea extract at 5 mug/mL in experiment group 3 (Rhodiola rosea reduction), and replacing the culture medium with a vascularization induction culture medium containing nano-reduced graphene oxide prepared by reducing rhodiola rosea extract at 5 mug/mL in comparison group (CTRL); after three days of treatment, the medium was replaced with vascular induction medium, the cells were collected by using Trizol at the time of 7 days, RNA was extracted, and the gene transcription level of the intracellular vascular differentiation index CD31 was detected.
The results are shown in Table 2 and FIG. 5.
TABLE 2
The effect of the nano-reduction graphene oxide prepared by reduction of the rhodiola rosea extract on the increase of the CD31 gene transcription level in Sheds is obviously higher than that of the nano-reduction graphene oxide prepared by reduction of obvious hydrazine hydrate or vitamin C. The nanometer reduced graphene oxide prepared by hydrazine hydrate reduction does not influence the differentiation of Sheds.
Detection example 3
The detection example detects the influence of nanometer reduced graphene oxide prepared by reduction of rhodiola rosea extract on endothelial differentiation of different mesenchymal stem cells. The detection method comprises the following steps:
inoculating DPSCs, sheds, BMSCs into cell culture plates according to 2.5X10 5 cells/hole respectively, culturing for 24h, replacing culture medium with vascularization induction culture medium containing 5 μg/mL nanometer reduced graphene oxide by experiment group (rGO), and replacing culture medium with vascularization induction culture medium without nanometer reduced graphene oxide by control group (CTRL); after three days of treatment, the medium was replaced with vascular induction medium, the cells were collected by using Trizol at the time of 7 days, RNA was extracted, and the gene transcription level of the intracellular vascular differentiation index CD31 was detected.
The results are shown in Table 3 and FIG. 6.
TABLE 2
The reduced graphene oxide prepared by reduction of the rhodiola rosea extract can selectively promote transcription of CD31 genes in Sheds, and has no influence on transcription of CD31 genes in DPSCs and BMSCs. The reduced graphene oxide prepared by the reduction of the rhodiola rosea extract can selectively induce and improve the angiopoiesis and differentiation capacity of Sheds.
Detection example 4
The present test example examined the angiogenesis promoting effect of the cell membrane of examples 1 to 3. The method comprises the following steps:
12 SD male rats weighing about 200-220 g are randomly divided into four groups, and are anesthetized by intraperitoneal injection, and are subjected to skin preparation and towel spreading in the right mandibular area, and are sterilized by iodophor alcohol. After local anesthesia in the operative field, an extraoral incision of about 2cm length was made along the lower edge of the right mandible, the subcutaneous fascia was separated layer by layer, the masseter fascia was severed, the periosteum was stripped, and the bone surface was exposed. The periodontal ligament and cementum on the root surface were thoroughly removed by carefully abrading the mandibular first molar mesial and buccal alveolar bones with a rapid turbine, creating a periodontal defect region of approximately 3mm x 2mm x 1mm in volume. The cells of example 1, example 2, example 3 or comparative example 1 were implanted in the defect with a large amount of physiological saline rinse, and the rat bone defect area of the control group was untreated. The bite muscle is reset, and the suture is sutured in layers. Antibiotics were injected continuously 3 days post-surgery to prevent infection. 2 weeks after surgery, contrast medium was injected systemically to mark microvascular formation before the experimental rats were sacrificed. After the experimental rat is sacrificed, the mandible of the experimental rat is taken down, decalcified step by step after fixation, and micro-CT three-dimensional reconstruction is performed, and the angiogenesis condition is analyzed through the area of the new blood vessel.
The results are shown in FIG. 7.
The cell sheets of examples 1 to 3 all had an angiogenesis promoting effect, and were able to promote a very significant increase in the area of the new blood vessel. Among them, the cell membrane of example 1 had significantly better angiogenesis promoting effect than the cell membranes of examples 2 to 3 and comparative example 1.
Detection example 5
This test example examined the bone defect repair effect of the cell membranes of examples 1 to 3 and comparative example 1. The method comprises the following steps:
12 SD male rats weighing about 200-220 g are randomly divided into four groups, and are anesthetized by intraperitoneal injection, and are subjected to skin preparation and towel spreading in the right mandibular area, and are sterilized by iodophor alcohol. After local anesthesia in the operative field, an extraoral incision of about 2cm length was made along the lower edge of the right mandible, the subcutaneous fascia was separated layer by layer, the masseter fascia was severed, the periosteum was stripped, and the bone surface was exposed. The periodontal ligament and cementum on the root surface were thoroughly removed by carefully abrading the mandibular first molar mesial and buccal alveolar bones with a rapid turbine, creating a periodontal defect region of approximately 3mm x 2mm x 1mm in volume. The cells of example 1, example 2, example 3 or comparative example 1 were irrigated with physiological saline and the defect was implanted without treatment in the rat bone defect area. The bite muscle is reset, and the suture is sutured in layers. Antibiotics were injected continuously 3 days post-surgery to prevent infection. 8 weeks after the operation, the mandible of the experimental rat is taken down, decalcified step by step after fixation, micro-CT three-dimensional reconstruction is carried out, and the osteogenesis is analyzed through the new bone formation area. After paraffin embedding and slicing of bone tissue, H & E staining is carried out, and the condition of new bone in the defect area is observed under a microscope.
The results are shown in fig. 8 and 9.
The cell membranes of examples 1 to 3 all had bone defect repair effects, and the new bone area was significantly increased. Among them, the cell membrane of example 1 was significantly superior to the cell membranes of examples 2 to 3 and comparative example 1 in bone defect repair effect.
The embodiments of the present invention have been described in detail with reference to the embodiments, but the present invention is not limited to the embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present invention.
Claims (10)
1. A cell patch, comprising:
A mesenchymal stem cell composite membrane comprising a angioblast-induced mesenchymal stem cell membrane; the mesenchymal stem cell membrane is pretreated by nano-reduced graphene oxide, and the nano-reduced graphene oxide is prepared by reducing graphene oxide by rhodiola rosea extract;
an endothelial cell composite membrane comprising an endothelial cell membrane; the endothelial cell composite membrane sheet is connected to the mesenchymal stem cell composite membrane sheet in a lamination manner.
2. The cell patch of claim 1, wherein the mesenchymal stem cells comprise at least one of deciduous dental pulp stem cells, and bone marrow mesenchymal stem cells;
And/or, the endothelial cells comprise at least one of umbilical vein endothelial cells and microvascular endothelial cells.
3. The cell membrane according to claim 1, wherein the preparation method of the nano-reduced graphene oxide comprises the following steps:
Mixing the rhodiola rosea extract with graphene oxide, and reacting to obtain the nano-reduced graphene oxide;
Preferably, the rhodiola rosea extract comprises at least one of rhodiola rosea alcohol extract and rhodiola rosea water extract.
4. The cell membrane of claim 1, wherein the number of layers of the mesenchymal stem cell membrane is 1-5;
and/or the number of layers of the endothelial cell membrane is 1-5.
5. Application of nanometer reduced graphene oxide prepared by reduction of radix Rhodiolae extract in preparation of product for promoting differentiation of deciduous tooth pulp stem cells into blood vessels or for non-disease diagnosis and treatment purpose.
6. The method for producing a cell membrane according to any one of claims 1 to 4, comprising the steps of:
S1, preparing the mesenchymal stem cell composite membrane:
Co-culturing the mesenchymal stem cells and the nano reduced graphene oxide in a angiogenesis induction culture medium, and transferring the culture medium to a mixed culture medium for culture to obtain the mesenchymal stem cell composite membrane;
the mixed culture medium comprises a angiopoiesis induction culture medium and a film forming culture medium;
s2, preparing the endothelial cell composite membrane sheet:
culturing endothelial cells in a film forming culture medium to obtain the endothelial cell composite membrane;
S3, preparing the cell membrane:
And (3) contacting and integrating the mesenchymal stem cell composite membrane with the endothelial cell composite membrane, and culturing in a vascularization induction culture medium to obtain the cell membrane.
7. The method of claim 6, wherein the angiogenesis-inducing medium comprises serum, endothelial growth factor, and a first basal medium;
And/or the film-forming medium comprises serum, L-glutamine, ascorbic acid, and a second basal medium.
8. The preparation method according to claim 6, wherein in the step S1, the concentration of the nano-reduced graphene oxide is 1-5 μg/mL;
and/or, in the step S1, the number of days of co-culture is 3-5 days.
9. The method according to claim 6, wherein in step S3, the integration time is 24-48 hours;
and/or, in the step S3, the culture time in the angiogenesis induction culture medium is 5-7 days.
10. Use of a cell membrane according to any one of claims 1 to 4 or a method of preparation according to any one of claims 6 to 9 for the preparation of a bone tissue engineering repair material or for the preparation of a medicament for promoting periodontal tissue regeneration.
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