CN115197904A - In-vitro evaluation model for bone repair material - Google Patents
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- CN115197904A CN115197904A CN202210711252.XA CN202210711252A CN115197904A CN 115197904 A CN115197904 A CN 115197904A CN 202210711252 A CN202210711252 A CN 202210711252A CN 115197904 A CN115197904 A CN 115197904A
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- C12N5/06—Animal cells or tissues; Human cells or tissues
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- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
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Abstract
The invention belongs to the technical field of medicine, discloses an in-vitro evaluation model of a bone repair material, and particularly discloses an in-vitro culture system of a multilayer bionic bone tissue, wherein the in-vitro culture system comprises a multilayer cell sheet laminated object and an osteogenesis induction culture solution, the multilayer cell sheet laminated object is composed of n bone marrow mesenchymal stem cell films, and n is more than or equal to 3. Compared with the traditional two-dimensional bone marrow mesenchymal stem cell culture system, the in-vitro culture system of the multilayer bionic bone tissue provided by the invention is closer to the three-dimensional shape of the in-vivo bone tissue microenvironment, ensures the mutual connection between compact osteoblasts and the interaction between the osteoblasts and a bone repair material, and is convenient for adding the bone repair material, so that the function and the characteristic of the bone repair material can be effectively evaluated, and the effectiveness of the in-vivo bone repair material can be more truly predicted.
Description
Technical Field
The invention belongs to the technical field of medicine, and particularly relates to an in-vitro evaluation model of a bone repair material.
Background
With the increase of bone defect cases, higher requirements are put on bone repair materials, and the bone repair materials also become one of the more popular research subjects in the biomedical material field. The quality and marketability of the bone repair material depends on its evaluation criteria. Due to species differences between animals and humans, animal models can only predict the reparability of bone repair materials in humans from large mammalian species. Therefore, it is important to establish a stable and reliable in vitro human primary bone repair model and to evaluate the repair properties of bone repair materials. However, in the process of ordinary two-dimensional in vitro culture of human primary mesenchymal stem cells, due to the lack of the three-dimensional structure of bone in vivo, intercellular interaction and the support of growth factors, the structural morphology of bone tissues in human body cannot be restored, thereby affecting the screening and evaluation of bone repair materials. Therefore, the long-term stable evaluation of the functions and properties of bone repair materials in vitro is an important consideration for establishing bone repair material evaluation models.
Human bone marrow mesenchymal stem cells (hbmscs) are a pluripotent stem cell that can differentiate into osteocytes, adipocytes and chondrocytes. It has strong proliferation and immunoregulation abilities, so it is widely used in tissue engineering, cell therapy and gene therapy.
At present, a three-dimensional in-vitro bionic tissue model system for maintaining osteogenesis function for a long time is not constructed by an hBMSC cell thin film lamination technology.
Disclosure of Invention
The first aspect of the invention aims to provide an in-vitro culture system of multilayer bionic bone tissues.
In a second aspect, the present invention is directed to a multilayered biomimetic bone tissue.
In a third aspect, the present invention provides a method for preparing the multilayered biomimetic bone tissue according to the second aspect.
The fourth aspect of the present invention is directed to the use of the in vitro culture system of the first aspect or the multilayered biomimetic bone tissue of the second aspect of the present invention for repairing a tissue or organ.
In a fifth aspect, the invention provides the use of the in vitro culture system of the first aspect or the multilayered biomimetic bone tissue of the second aspect for tissue regeneration.
The sixth aspect of the invention aims to provide an in vitro evaluation model of bone repair materials.
An object of the seventh aspect of the present invention is to provide a method for evaluating a bone repair material.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention provides an in-vitro culture system of multilayer bionic bone tissues, which comprises a multilayer cell sheet laminated object and an osteogenesis induction culture solution, wherein the multilayer cell sheet laminated object is composed of n bone marrow mesenchymal stem cell films, and n is more than or equal to 3.
Preferably, the preparation method of the mesenchymal stem cell film comprises the following steps: inoculating the bone marrow mesenchymal stem cells into a culture container, culturing and stripping to obtain the bone marrow mesenchymal stem cell film.
Preferably, the inoculation number of the mesenchymal stem cells is 30-50 ten thousand; preferably 30 to 40 ten thousand; more preferably 40 ten thousand.
Preferably, the culture condition is culture at 30-39 ℃ for 4-7 days; further culturing for 4-7 days at 35-37 ℃; most preferably 37 ℃ and 5% CO 2 The culture was carried out at concentration for 4 days.
Preferably, when the culture container is a common culture dish, a ring-shaped material is attached to the interior of the culture dish before the mesenchymal stem cells are inoculated; the annular material is prepared from high-molecular hydrogel or silica gel; the annular material can be quickly adhered to the culture container, has no leakage phenomenon when being tightly assembled together, and can be freely detached.
Preferably, the method of peeling is to lightly blow the edge of the culture dish with a pipette.
Preferably, the in vitro culture system further comprises a culture solution.
Preferably, the culture solution comprises a DMEM basic culture medium, 0.1-0.2 mu M dexamethasone, 0.05-0.1 g/L ascorbic acid, 20-30 g/L Ficoll400, 1-3 w/v% ITS, 10-20 v/v% fetal calf serum and 1-3 w/v% streptomycin.
Preferably, the mesenchymal stem cells are primary cell pluripotent stem cells.
Preferably, the source of the mesenchymal stem cells is human.
In a second aspect of the present invention, there is provided a multilayered biomimetic bone tissue cultured from the in vitro culture system for a multilayered biomimetic bone tissue according to the first aspect of the present invention.
In a third aspect of the present invention, there is provided a method for producing a multilayered biomimetic bone tissue according to the second aspect of the present invention, comprising the steps of:
(1) Placing the bone marrow mesenchymal stem cell film in a culture container for culture;
(2) Overlaying the bone marrow mesenchymal stem cell film on the bone marrow mesenchymal stem cell film in the step (1) and culturing;
(3) Repeating the step (2) until obtaining a bone marrow mesenchymal stem cell film with n layers, wherein n is more than or equal to 3, and obtaining a multilayer cell sheet lamination substance;
(4) Adding osteogenic induction culture solution, and culturing to obtain multilayer bionic bone tissue.
Preferably, the osteogenesis induction medium comprises ascorbic acid, sodium beta-glycerophosphate and dexamethasone.
Preferably, the culture conditions in step (1) and step (2) are 30 to 39 ℃ and 3% to 7% CO 2 Culturing for 25-35 min; further, 35 to 37 ℃ and 5 to 7% of CO 2 Culturing for 30-35 min; further 37 ℃ and 5% 2 The culture is carried out at a concentration for 30min.
Preferably, the culturing in step (4) is carried out under conditions of 30 to 37 ℃ and 3 to 7% CO 2 Culturing for 3-14 days; further, 35 to 37 ℃ and 5 to 7% of CO 2 Culturing for 3-7 days; further 35 ℃ and 5% CO 2 The culture was concentrated for 7 days.
In a fourth aspect of the invention, there is provided the use of the in vitro culture system of the first aspect of the invention or the multilayered biomimetic bone tissue of the second aspect of the invention for repairing a tissue organ.
In a fifth aspect of the invention, there is provided the use of the in vitro culture system of the first aspect of the invention or the multilayered biomimetic bone tissue of the second aspect of the invention for tissue regeneration.
In a sixth aspect of the invention, there is provided a model for in vitro evaluation of a bone repair material, comprising a multilayered biomimetic bone tissue according to the second aspect of the invention.
Preferably, the multilayer bionic bone tissue is prepared into a 3D structure with a hollow-out middle part.
In a seventh aspect of the present invention, there is provided an evaluation method of a bone repair material, the evaluation method comprising the steps of: mixing and culturing the bone repair material and the multilayer bionic bone tissue of the second aspect of the invention, and detecting the expression level of osteogenic protein or osteogenic gene; or
The method is carried out by adopting the bone repair material in-vitro evaluation model of the sixth aspect of the invention.
Preferably, the culture conditions are 30 to 37 ℃ and 3 to 7% of CO 2 Culturing for 3-14 days; further, 35 to 37 ℃ and 5 to 7% of CO 2 Culturing for 3-7 days; further 35 ℃ and 5% CO 2 The culture was concentrated for 7 days.
Preferably, the osteogenic protein comprises RUNX, OSX, OCN and OPN.
The invention has the beneficial effects that:
compared with a traditional two-dimensional bone mesenchymal stem cell culture system, the in-vitro culture system of the multilayer bionic bone tissue provided by the invention is closer to the three-dimensional form of a microenvironment of the in-vivo bone tissue, ensures the mutual connection between compact osteoblasts and the interaction between the osteoblasts and a bone repair material, and is convenient for adding the bone repair material, so that the function and the characteristic of the bone repair material can be effectively evaluated, and the effectiveness of the in-vivo bone repair material can be more truly predicted.
Compared with the existing artificial bone material, the multi-layer bionic bone tissue of the mesenchymal stem cells provided by the invention does not need a biological material as a scaffold for bearing, is purely composed of homologous cells, reduces the pollution caused by immune rejection and scaffold degradation, forms a three-dimensional bionic bone tissue in vitro, and has important significance for repairing bone defects and regenerating tissues.
The multi-layer bionic bone tissue of the mesenchymal stem cells is obtained by culturing and stripping the hBMSC cell film and successfully laminating, the multi-layer bionic bone tissue is completely composed of cells and extracellular matrix generated by the cells, does not contain any exogenous scaffold material, is fundamentally different from other three-dimensional bionic tissues constructed by hydrogel or the scaffold material, can highly reduce the microenvironment of the bone tissue in vivo, and is more suitable for long-term and accurate evaluation of the osteogenic property of a bone repair material.
Drawings
FIG. 1 is a schematic diagram of the construction of a multilayer bionic bone tissue of human mesenchymal stem cells.
FIG. 2 is a graph showing the expression results of osteogenesis-related proteins and related genes in a multilayered bionic bone tissue of human mesenchymal stem cells; a is a graph of expression result of bone formation related protein of the multilayer bionic bone tissue of the human mesenchymal stem cells, B is a graph of expression result of bone formation related gene of the multilayer bionic bone tissue of the human mesenchymal stem cells, and p is less than 0.05 and less than 0.01.
Fig. 3 is a schematic diagram of the construction of an in vitro evaluation model of a bone repair material.
FIG. 4 is a result diagram of three bone repair materials whose osteogenesis inducing properties are evaluated by an in vitro bone tissue bionic evaluation model; wherein A is a real image of three bone repair materials in an in vitro bone tissue bionic evaluation model, and B is an expression result image of osteogenesis related protein of a human bone marrow mesenchymal stem cell multilayer bionic bone tissue added with the bone repair materials.
Fig. 5 is a graph showing the expression results of osteogenesis-related genes in a multilayered human mesenchymal stem cell-derived bone tissue to which a bone repair material was added, wherein p is <0.05, p is < 0.01, and p is < 0.005.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
The materials, reagents and the like used in the present examples are commercially available materials and reagents unless otherwise specified.
Example 1 construction of multilayer Bionical bone tissue of human mesenchymal Stem cell
Expanding and culturing human bone marrow mesenchymal stem cells (hBMSC): human bone marrow mesenchymal stem cellFriendly donation from oral medical college of Zhongshan university), was routinely cultured using a-MEM (purchased from Corning) medium containing 10v/v% FBS, trypsinized, and cultured at 75cm 2 Amplifying and culturing cells in culture flask, and placing in 5% CO at 37 deg.C 2 Culturing in an incubator until the cells grow to 90%. Well-grown hBMSC was made into hBMSC suspension using hBMSC thin film medium (DMEM (available from Corning) + 0.1. Mu.M dexamethasone (available from Sigma Aldrich) +0.05g/L ascorbic acid (available from Solarbio) +25g/L Ficoll400 (available from Cytiva) +1w/v% ITS cell culture supplement (available from Oricell), 10v/v% FBS, 1w/v% streptomycin).
Attaching a silica gel ring with an inner diameter of 15.5mm to a 35mm common petri dish, inoculating a suspension of hBMSC (containing 40 ten thousand cells) to the center of the silica gel ring (or directly inoculating the suspension of hBMSC to a common 24-well plate), standing at 37 deg.C, and 5% CO 2 Culturing in an incubator for 4d; replacing the hBMSC thin film forming culture medium every 2 days to form a bone marrow mesenchymal stem cell thin film; taking off the silica gel ring, and then gently blowing and beating the silica gel ring along the side wall by using a 1mL pipette to peel off the mesenchymal stem cell film (when a common 24-pore plate is used, gently blowing and beating the silica gel ring along the side wall by using the 1mL pipette directly to peel off the mesenchymal stem cell film); sucking the culture medium in the stripped human mesenchymal stem cell film, placing in a 35mm culture dish, and culturing at 37 deg.C with 5% CO 2 Placing in a culture box for 30min to adhere the human mesenchymal stem cell film to a culture dish; overlaying the second piece of exfoliated human mesenchymal stem cell membrane on the first piece, trying to completely overlap, blotting the surrounding medium, and incubating at 37 deg.C and 5% CO 2 Placing in an incubator for 30min to ensure that the two human mesenchymal stem cell films are fully bonded; further overlapping a third piece of peeled human mesenchymal stem cell thin film on the second piece, trying to completely overlap, blotting the surrounding culture medium, and drying at 37 deg.C and 5% CO 2 Placing in an incubator for 30min to ensure that three sheets of human mesenchymal stem cell films are fully bonded; then, 2mL of osteogenic culture solution (purchased from Cyagen oricell) is added into a culture dish for culturing the laminated tissue to form a human mesenchymal stem cell thin film multilayer bionic bone tissue culture system; placing the multilayer bionic bone tissue culture system at 37 deg.C and 5% CO 2 Culturing in a culture box to obtain the multilayer bionic bone tissue of the human mesenchymal stem cells.
Fig. 1 shows a schematic diagram of construction of a multilayer bionic bone tissue of human bone marrow mesenchymal stem cells.
Example 2 construction of in vitro evaluation model of bone repair Material
Expanding and culturing the human bone marrow mesenchymal stem cells: human mesenchymal stem cells (kindly donated by oral medical institute of Zhongshan university) were routinely cultured using a-MEM (available from Corning) medium containing 10v/v% FBS, trypsinized, and cultured at 75cm 2 Amplifying and culturing cells in a culture flask, and placing in 5% CO at 37 deg.C 2 Culturing in an incubator until the cells grow to 90%. Well-grown hBMSC human mesenchymal stem cells were made into hBMSC suspension using hBMSC thin film medium (DMEM (from Corning) + 0.1. Mu.M dexamethasone (from Sigma Aldrich) +0.05g/L ascorbic acid (from Solarbio) +25g/L Ficoll400 (from Cytiva) +1w/v% ITS cell culture supplement (from Oricell), 10v/v% FBS, 1w/v% streptomycin).
A circular sealing film with a diameter of 5mm was attached to the center of each well of a 24-well plate (or to the center of a 35-mm common petri dish to which a 15.5mm ring-shaped material had been attached), and the hBMSC suspension (40 ten thousand) was inoculated into the well of the 24-well plate (center of the silica gel ring), and the mixture was placed at 37 ℃ and 5% CO 2 Culturing for 4d in an incubator to form a mesenchymal stem cell film with a hole in the middle (a circular hole with the diameter of 5 mm). Taking off the silica gel ring, and then gently blowing and beating the silica gel ring along the side wall by using a 1mL pipette to peel off the mesenchymal stem cell film (if the silica gel ring is a 24-hole plate, gently blowing and beating the silica gel ring along the side wall by using a 1mL pipette to peel off the mesenchymal stem cell film); sucking the culture medium in the stripped human mesenchymal stem cell film, placing in a 35mm culture dish, and culturing at 37 deg.C with 5% CO 2 Placing in a culture box for 30min to adhere the human mesenchymal stem cell film to a culture dish; overlaying the second piece of exfoliated human mesenchymal stem cell membrane on the first piece, trying to completely overlap, blotting the surrounding medium, and incubating at 37 deg.C and 5% CO 2 Placing in an incubator for 30min to ensure that the two human mesenchymal stem cell films are fully bonded; further stripping the third piece of the human boneOverlaying the mesenchymal stem cell membrane on the second sheet, collecting completely overlapped mesenchymal stem cell membrane, blotting the surrounding culture medium, and culturing at 37 deg.C under 5% CO 2 Placing in the incubator for 30min to make three sheets of human bone marrow mesenchymal stem cell films fully bonded to obtain the human bone marrow mesenchymal stem cell film multilayer bionic bone tissue with a hole in the middle (a circular hole with the diameter of 5 mm).
Placing 2mg of osteogenic material (such as hydroxyapatite bioceramic (Beijing Yihua Jiankou, inc., ZC-J basic type, production lot 200911), self-curing calcium phosphate bone repair material (Guangzhou Xifu medical equipment, inc., production lot 20180504) or bone cement (main component: polymethyl methacrylate)) in 5mm pores of the human mesenchymal stem cell thin film multilayer bionic bone tissue, and covering a collagen film (Nuo Yi Mei (Suzhou) medical science and technology, inc., production lot AGM 190401) thereon to prevent the osteogenic material from being washed away by osteogenic culture solution; adding 1mL of osteogenic culture solution into the culture dish to form a human bone marrow mesenchymal stem cell thin film multilayer co-culture system; subjecting the multi-layer CO-culture system to 5% CO at 37 deg.C 2 And culturing in a culture box to obtain an in-vitro evaluation model of the bone repair material.
A schematic diagram of the construction of an in vitro evaluation model of bone repair material is shown in fig. 3.
Effects of the embodiment
1. Osteogenic characteristic verification of multilayer bionic bone tissue of human bone marrow mesenchymal stem cells
By using the method for constructing a multilayered bionic bone tissue of human mesenchymal stem cells in example 1, a multilayered bionic bone tissue of human mesenchymal stem cells, which is induced by osteogenesis with an osteogenic culture solution for 0d, 7d, or 14d, is obtained. Collecting the cells of the two human bone marrow mesenchymal stem cells in the multilayer bionic bone tissue, extracting total RNA and total protein of the cells, verifying the expression of bone formation related genes (RUNX 2: participating in the initiation of osteogenic differentiation, OSX: regulating and controlling the mineralization of bone, OCN: osteocalcin and OPN: osteopontin, and beta-actin as an internal reference gene) by using qPCR, and verifying the expression of the bone formation related proteins by using Western blot.
(1)qPCR
The method for extracting the total RNA of the cells comprises the following steps: preparing a gun head without RNase and an EP tube in advance, taking cells, transferring the cells into a 1.5mL EP tube, centrifuging for 5min at 500g, discarding supernatant, washing for 2 times by PBS, and reserving precipitated cells; 1mL Trizol (from Ambion) and 200. Mu.L chloroform were added, shaken, and ice-cooled on ice for 30min; centrifuging at 12000g for 15min at 4 deg.C, sucking 500 μ L of supernatant, adding 500 μ L of isopropanol (from Macklin), and bathing in ice water for 30min; centrifuging at 12000g for 15min to obtain white precipitate as RNA; adding 700 μ L75% ethanol, mixing, centrifuging at 4 deg.C and 12000g for 7min; the supernatant was discarded, air-dried, and then added with RNA-free water to measure the concentration.
Reverse transcription of the extracted RNA to cDNA according to the protocol of the reverse transcription kit (purchased from Takara); the qPCR assay was then performed, with 3 replicates per sample. 20 μ L of the reaction system respectively included: 1. Mu.L of forward primer (10. Mu.M), 1. Mu.L of reverse primer (10. Mu.M), 1. Mu.L of cDNA template, 7. Mu.L of ultrapure water, and 10. Mu.L of mix (Genstar, cat # A311-10). The components are shaken and mixed uniformly in a dark place, then added into a qPCR tube, covered with a cover, centrifuged to remove air bubbles, and placed into a fluorescence quantitative PCR instrument (Thermo Fisher). Setting reaction conditions: pre-denaturation at 95 ℃ for 30s; denaturation at 95 ℃ for 0.05s, renaturation at 60 ℃ for 30s,45 cycles. The primer sequences of the above primers are shown in Table 1.
TABLE 2 primer sequences
(2)Western blot
Cell lysate was added to the above cells according to the instructions of the cell lysate (purchased from Beyotime) and the cells were lysed on ice for 30min; centrifuging at 14000g for 30min to obtain supernatant and obtain protein of each cell; the content of the resulting protein was determined using BCA protein assay kit (Thermo). Preparing 10% separation gel, standing for 25min, and standing until the separation gel is solidified; preparing 5% concentrated glue, standing for 25min, and vertically pulling out the comb after concentration and solidification. Subsequently, each group of proteins was separated by SDS-PAGE (86V running out of the gel for about 30min, then 200V electrophoresis to separate the bands), and the protein samples on the gel were transferred to a PVDF membrane (purchased from lmmobilon, activated by immersing the PVDF membrane in methanol before transfer) (110 mA transfer overnight), and the transferred PVDF membrane was immersed in 5% skimmed milk powder and sealed at room temperature for 1h; the PVDF membrane was then immersed in primary antibody (RUNX 2, OSX, OCN, OPN, or GAPDH, where RUNX2 was purchased from Cell Signaling, cat # 12556, OSX, OCN, OPN, and GAPDH were purchased from Abcam, cat # ab209484, ab133612, ab214050, ab181602, respectively) working solution (formulated with primary antibody diluent (Beyotime, cat # AZ 100) solution 1, 1000) and incubated at room temperature for 1-2 h; taking out the PVDF membrane, and washing the PVDF membrane with TBST for three times, 10min each time; PVDF membranes were immersed in a secondary antibody (anti-rabbit antibody, purchased from Beyotime, cat No. a 0208) working solution (diluted with milk at a dilution ratio of 1 to 3000) at room temperature and incubated for 1h; taking out the PVDF membrane, and washing the PVDF membrane for three times by TBST, wherein each time is 10min; the image was developed on a developing apparatus (Tanon 5200 Multi).
By detecting the osteogenesis related genes and related proteins of the human mesenchymal stem cell multilayer bionic bone tissue with osteogenesis induction of 7d and 14d, the result shows that the expression level of the osteogenesis related proteins and genes in the human mesenchymal stem cell multilayer bionic bone tissue is remarkably increased (p is less than 0.05) along with the prolongation of the osteogenesis induction time (figure 2), and the obtained human mesenchymal stem cell multilayer bionic bone tissue has better bone performance.
2. Evaluation of osteoinductive Properties of bone repair Material
Three bone repair materials (hydroxyapatite bioceramic, ZC-J basic type, production lot 200911, beijing yihua jiaoqiao limited company), self-setting calcium phosphate bone repair material (canthoff medical equipment limited, production lot 20180504, guangzhou) and bone cement (main component: polymethylmethacrylate)) were each subjected to the in vitro evaluation model of the bone repair material of example 2 to evaluate their bone induction characteristics. Collecting human bone marrow mesenchymal stem cells which are induced by an osteogenesis induction culture medium for 0d, 14d and 28d, extracting total RNA and total protein of the cells, verifying expression of osteogenesis related genes by using qPCR (quantitative polymerase chain reaction) and expression of osteogenesis related proteins by using Western blot (the specific experimental process is verified by the osteogenesis characteristics of multilayer bionic bone tissues of the human bone marrow mesenchymal stem cells), and recording the compatibility of bone repair materials and the bionic bone tissue model in each week.
As shown in fig. 4 and 5, after the three bone repair materials were added to the in vitro evaluation model of the bone repair material, the in vitro evaluation model was cultured in 14d and 28d osteogenesis induction media, and the in vitro evaluation model was stable in structure, and it was observed that the bone repair material and the human bone marrow mesenchymal stem cell multilayer bionic bone tissue had better fusion property with time (a in fig. 4); from day 0 until day 28, the multilayer biomimetic bone tissue of human bone marrow mesenchymal stem cells was osteo-induced, and the osteogenesis-related proteins exhibited a state maintained after a gradual increase, showing no significant decrease (B in fig. 4). It can also be seen from the gene data by PCR that the osteogenesis-related gene shows a stable maintenance state after the increase, and it can be observed that the osteogenic repair property of the bone cement and the self-setting calcium phosphate is superior to that of the hydroxyapatite bioceramic (fig. 5). In conclusion, it is shown that the multilayer bionic bone tissue is beneficial to reproducing the three-dimensional shape of the bone tissue in vitro, and the in vitro evaluation model of the bone repair material in the embodiment 2 can effectively evaluate the repair characteristics and effectiveness of the bone repair material.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and features of the embodiments may be combined with each other without conflict.
SEQUENCE LISTING
<110> institute of biological and medical engineering, academy of sciences of Guangdong province
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Claims (10)
1. The in-vitro culture system of the multilayer bionic bone tissue is characterized by comprising a multilayer cell sheet laminated object and osteogenesis induction culture solution, wherein the multilayer cell sheet laminated object is composed of n mesenchymal stem cell films, and n is more than or equal to 3.
2. The in vitro culture system of claim 1, wherein the preparation method of the mesenchymal stem cell membrane comprises the following steps: inoculating the mesenchymal stem cells into a culture container, culturing and stripping to obtain the mesenchymal stem cell film; preferably, the culture conditions are 30 to 39 ℃ and 3 to 7% of CO 2 Culturing for 4-7 days under the concentration.
3. A multilayered simulated bone tissue cultured using the in vitro culture system for the multilayered simulated bone tissue according to claim 1 or 2.
4. The method for preparing a multilayered biomimetic bone tissue according to claim 3, comprising the preparation steps of:
(1) Placing the bone marrow mesenchymal stem cell film in a culture container for culture;
(2) Overlaying the bone marrow mesenchymal stem cell film on the bone marrow mesenchymal stem cell film in the step (1) and culturing;
(3) Repeating the step (2) until obtaining a bone marrow mesenchymal stem cell film with n layers, wherein n is more than or equal to 3, and obtaining a multilayer cell sheet lamination substance;
(4) Adding osteogenic induction culture solution, and culturing to obtain multilayer bionic bone tissue.
5. The method of claim 4, wherein the osteogenesis inducing medium comprises ascorbic acid, sodium beta-glycerophosphate, and dexamethasone.
6. The process according to claim 5, wherein the conditions for the culture in the steps (1) and (2) are 30 to 39 ℃ and 3 to 7% by weight of CO 2 Culturing for 25-35 min; preferably, the culturing in step (4) is carried out under conditions of 30 to 37 ℃ and 3 to 7% CO 2 Culturing at the concentration for 3-14 days.
7. Use of the in vitro culture system of claim 1 or 2 or the multilayered biomimetic bone tissue of claim 3 for repairing soft tissue organs.
8. Use of the in vitro culture system of claim 1 or 2 or the multilayered biomimetic bone tissue of claim 3 for tissue regeneration.
9. An in vitro evaluation model of a bone repair material comprising the multilayered biomimetic bone tissue of claim 3.
10. An evaluation method of a bone repair material, characterized in that the evaluation method comprises the steps of: culturing a bone repair material in admixture with the multilayered biomimetic bone tissue according to claim 3, and detecting an expression amount of osteogenic protein or osteogenic gene;
or
Using the bone repair material in vitro evaluation model of claim 9.
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