CN111282020A - Matrix-dependent tissue engineering bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells and preparation method thereof - Google Patents
Matrix-dependent tissue engineering bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells and preparation method thereof Download PDFInfo
- Publication number
- CN111282020A CN111282020A CN202010091980.6A CN202010091980A CN111282020A CN 111282020 A CN111282020 A CN 111282020A CN 202010091980 A CN202010091980 A CN 202010091980A CN 111282020 A CN111282020 A CN 111282020A
- Authority
- CN
- China
- Prior art keywords
- cells
- mesenchymal stem
- bone
- matrix
- tissue engineering
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—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
- 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
- A61L27/3804—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 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—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
- A61L27/3604—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 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3608—Bone, e.g. demineralised bone matrix [DBM], bone powder
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—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
- A61L27/3641—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 characterised by the site of application in the body
- A61L27/3645—Connective tissue
- A61L27/365—Bones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—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
- 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
- A61L27/3804—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 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3808—Endothelial cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/56—Porous materials, e.g. foams or sponges
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/069—Vascular Endothelial cells
- C12N5/0692—Stem cells; Progenitor cells; Precursor cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2531/00—Microcarriers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Cell Biology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Dermatology (AREA)
- Medicinal Chemistry (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Transplantation (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Developmental Biology & Embryology (AREA)
- Botany (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Urology & Nephrology (AREA)
- Microbiology (AREA)
- Vascular Medicine (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Rheumatology (AREA)
- Dispersion Chemistry (AREA)
- Materials For Medical Uses (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a preparation method of a matrix-dependent tissue engineering bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells, which comprises the steps of planting the mesenchymal stem cells and the endothelial progenitor cells on a porous bone scaffold material according to the proportion of 1:1 to construct a tissue engineering complex, adding an osteogenesis induction culture solution in vitro to continue culturing for 13-15 days, freezing at-70-80 ℃ for 48-72 hours, freeze-drying for 24-30 hours, and freezing and storing for 1-3 months to obtain the matrix-dependent tissue engineering bone. In vitro research results show that the matrix-dependent tissue engineering bone constructed by the method based on the mesenchymal stem cells/endothelial progenitor cells as seed cells can remarkably promote endothelial progenitor cell migration, scratch repair and tube cavity formation in vitro and promote osteogenic differentiation of the mesenchymal stem cells; in vivo experiments show that the bone repair agent can remarkably promote the formation of new bones and successfully repair bone defects.
Description
Technical Field
The invention belongs to the technical field of tissue engineering, and relates to a matrix-dependent tissue engineering bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells and a preparation method thereof.
Background
The treatment of large bone defects caused by trauma, tumor, infection and the like is one of the difficulties of clinical treatment. At present, autologous bone transplantation still serves as the 'gold standard' of clinical bone transplantation, but has the limitations of small bone taking amount, trauma of bone taking areas and the like. Tissue Engineering Bone (TEB) is a hot spot of recent research and has achieved good clinical effects, but also has problems of living cell dependence, autologous cell dependence, and harsh storage and transportation conditions.
In order to solve the above problems, we established a bone matrix material containing a plurality of proteins secreted by umbilical cord mesenchymal stem cells and a preparation method (patent ZL 201310449152.5) in advance, and proposed a construction concept and system of "matrix-dependent tissue engineering bone (ECM-TEB)". Namely, ECM-TEB is constructed and obtained by removing the activity of seed cells and retaining active proteins such as various cytokines secreted by the cells. Although the cell activity is removed in the technical mode, the cell factors and matrix proteins which are wrapped on the scaffold material layer by layer in an autocrine mode are still retained, and the cell factors wrapped in the matrix are slowly released to the injury and participate in bone reconstruction under the action of enzyme after in vivo transplantation, so that the bioactive proteins loaded and released by the decellularized ECM-TEB meet the requirements of physiological environment. The basic construction strategy is as follows: compounding Mesenchymal Stem Cells (MSC) with a bone scaffold material, culturing for 14 days, freezing the cell-scaffold compound at-80 ℃ for 48 hours, freeze-drying for 24 hours, and freezing and storing for 3 months to form the ECM-TEB with the MSC as a seed cell and obviously reducing immunogenicity. 80% of active protein in the strategy is reserved, the defects of long time and high consumption of individualized tissue engineering bone and influence of the autologous stem cell state of a patient are effectively overcome, the preparation is simple and easy to implement, the effect is obvious, the popularization is convenient, and a new thought is provided for the research of tissue engineering.
The dynamic balance of bone reconstruction is realized by the mutual cooperation of various cells in bone tissues, so that the microenvironment for in-vivo osteogenesis can be more truly simulated if different types of cells can be established for co-culture in the construction of ECM-TEB. The MSC ECM-TEB constructed by taking MSC as a single seed cell shows good application prospect in repairing small-sized bone defects, but poor vascularization of defect parts is often caused due to lack of perfect vascular systems in repairing large bone defects, and the clinical application range of tissue engineering bone is limited. With the development of regenerative medicine, ECM-TEB with angiogenesis potential has great significance for repairing large bone defects. Endothelial Progenitor Cells (EPCs) can be differentiated into vascular endothelial cells to generate blood vessels, and can promote MSCs to home and form bones under a specific microenvironment, so that the invention has excellent clinical significance in introducing EPC as seed cells while MSC is used as osteoblast differentiated seed cells, and exploring MSC/EPC matrix-dependent tissue engineering bones constructed by the 'composite' seed cells.
At present, no report is found about constructing a matrix-dependent tissue engineering bone by using endothelial progenitor cells as one of seed cells and treating bone defects.
Disclosure of Invention
In view of the above, the present invention aims to establish a novel tissue engineering bone construction and introduce EPC as one of seed cells when a large tissue engineering bone is applied to repair a large bone defect, so as to solve the problems that it is difficult to construct a matrix-dependent tissue engineering bone using MSC as a single seed cell source, so that it is difficult to more truly simulate an in vivo osteogenesis microenvironment, and poor vascularization of a defect part is easily caused. The invention provides the following technical scheme:
1. the preparation method of the matrix-dependent tissue engineering bone constructed by taking the mesenchymal stem cells/endothelial progenitor cells as seed cells comprises the steps of planting the mesenchymal stem cells and the endothelial progenitor cells on a porous bone scaffold material according to the ratio of 1:1 to construct a tissue engineering complex, adding an osteogenic induction culture solution in vitro to continue culturing for 13-15 days, freezing at-70-80 ℃ for 48-72 hours, freeze-drying for 24-30 hours, and freezing and storing for 1-3 months to obtain the matrix-dependent tissue engineering bone.
Further, the mesenchymal stem cell is a bone marrow mesenchymal stem cell, a peripheral blood mesenchymal stem cell, a cord blood mesenchymal stem cell, an adipose mesenchymal stem cell or an umbilical cord mesenchymal stem cell.
Further, the mesenchymal stem cell is an umbilical cord mesenchymal stem cell.
Further, the porous bone scaffold material is a synthetic bone scaffold material, a decellularized bone matrix or a decalcified bone matrix.
Further, the porous bone scaffold material is a decalcified bone matrix.
Further, the preparation steps are as follows: soaking the decalcified bone matrix scaffold material in a culture medium for 48 hours, and adjusting the pH value to 7.2; taking the second-generation mesenchymal stem cells in the exponential growth period, washing the second-generation mesenchymal stem cells by PBS, incubating the second-generation mesenchymal stem cells for 1 hour by using a 0.1 wt% type I collagenase solution, adding pancreatin and a 0.02 wt% EDTA solution for digestion for 3-5 minutes, washing and re-suspending the second-generation mesenchymal stem cells by using a DMEM medium containing 10 wt% fetal calf serum, and mixing and re-suspending the second-generation mesenchymal stem cells and endothelial progenitor cells according to a ratio of 1: 1; inoculating cells to the decalcified bone matrix scaffold material after the treatment, so that the cell suspension just infiltrates the scaffold material and does not overflow out of the scaffold material; turning over the bottom surface of the decalcified bone matrix scaffold material to become a top surface after 3 hours under the aseptic operation condition, and inoculating cell suspension by the same method as the method; the cell scaffold has a regular size of 0.5cm × 0.5cm × 0.3cm, and the total number of planted cells is about 106(ii) a After 3 hours, DMEM medium was added to just over the top surface of the decalcified bone matrix scaffold material, and then 5% CO was added at 37 deg.C2Culturing under the condition; after 24 hours, carrying out osteogenic differentiation induction culture on the cells in vitro for 13-15 days; freezing for 48-72 hours at-70-80 ℃, freeze-drying for 24-30 hours, and freezing and storing for 1-3 months to obviously reduce immunogenicity, so as to form the matrix-dependent tissue engineering bone constructed by taking the mesenchymal stem cells/endothelial progenitor cells as seed cells.
2. The matrix-dependent tissue engineering bone constructed by the preparation method based on the mesenchymal stem cells/endothelial progenitor cells as seed cells.
The invention has the beneficial effects that: the invention establishes the matrix-dependent tissue engineering bone constructed based on the mesenchymal stem cells/endothelial progenitor cells as seed cells for the first time, not only widens the construction mode of the matrix-dependent tissue engineering bone, but also can better solve the vascularization bionic microenvironment. The results of in vivo studies show that the matrix-dependent tissue engineering bone constructed by the method based on the mesenchymal stem cells/endothelial progenitor cells as seed cells can successfully repair bone defects in vivo, has remarkably enhanced advantages in cell recruitment, angiogenesis and new bone generation, and effectively solves the problems of blood vessel microenvironment and the like of a bone grafting bed, so that the method has good application prospects in tissue engineering bone construction and bone defect repair.
Drawings
In order to make the object, technical solution and advantages of the present invention more clear, the present invention provides the following drawings for illustration.
Fig. 1 is a schematic diagram of a strategy for constructing a matrix-dependent tissue-engineered bone based on mesenchymal stem cells/endothelial progenitor cells as seed cells.
Fig. 2 is a graph showing the experimental results of the protein leaching solution of the matrix-dependent tissue engineering bone constructed based on the mesenchymal stem cells/endothelial progenitor cells as seed cells on the EPC scratch repair in vitro.
Fig. 3 is a graph showing the results of in vitro migration experiments of matrix-dependent tissue engineered bone constructed based on mesenchymal stem/endothelial progenitor cells as seed cells to EPC.
Fig. 4 is a graph showing the experimental result of the protein leaching solution of the matrix-dependent tissue engineering bone constructed based on the mesenchymal stem cells/endothelial progenitor cells as seed cells on the osteogenic differentiation of MSC.
Fig. 5 is a graph showing the experimental results of protein leaching solution of matrix-dependent tissue engineering bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells on the formation of EPC lumen in vitro.
Fig. 6 is a graph of the results of rat femoral defect repair experiments performed on the matrix-dependent tissue engineering bone constructed based on the mesenchymal stem cells/endothelial progenitor cells as seed cells, and the results of Micro-CT examination and Masson staining detection 2 months after planting.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The experimental procedures, in which specific conditions are not specified in the preferred examples, are generally carried out according to conventional conditions or according to conditions recommended by the reagent manufacturers.
Example 1 construction of a matrix-dependent tissue engineered bone based on mesenchymal Stem cells/endothelial progenitor cells as seed cells
1. Culture of endothelial progenitor cells
Taking a 15mL centrifuge tube, adding 5mL of marrow lymphocyte separation liquid, carefully sucking the marrow single cell suspension on the liquid surface of the separation liquid, 400-500g, and centrifuging at low temperature for 30 min. Carefully sucking out the second lymphocyte layer, washing cells with a washing solution, inoculating into a culture dish paved with matrigel, adding EGM2-MV culture medium for culture, and replacing fresh culture solution for the first time after 3 days. After 7 days of cell culture, washing the cells with PBS for 2 times, adding Dil-ac-LDL (10 mug/mL), incubating the adherent cells for 4 hours in a dark place at 37 ℃, washing the cells for 3 times with PBS, fixing the cells for 15-20 min with 5% paraformaldehyde, washing the cells for 3 times with PBS, adding FITC-UEA-1(10 mug/mL) to incubate for 1 hour at 37 ℃, rinsing the cells for 3 times with PBS, and observing the capability of the cells to take in Dil-ac-LDL and combine with FITC-UEA-1 under a fluorescence microscope for identification. EPCs primary cells appear circular, adhere within 72 hours, and are predominantly fusiform. The DIL-AC-LDL and FITC-UEA-1 double-positive cells are identified as endothelial progenitor cells by a double-swallow experiment.
2. Culture of umbilical cord MSCs
Cutting off artery and vein of umbilical cord of healthy fetus, and cutting into pieces; culturing with tissue block, and digesting umbilical cord with type I collagenase; cleaning the tissue blocks by phosphate buffer solution, transferring the tissue blocks into a culture bottle, and adding a culture medium for adherent culture; culturing in 37 deg.C 5% CO2 incubator, changing liquid every 3 days, observing cell growth and morphological characteristics under inverted microscope, and inoculating cellsNear fusion was digested with 0.25% trypsin and passaged at a 1: 2 ratio. When the cultured bone marrow MSCs are fused to about 80%, 1:1, 2.5g/L trypsin and 0.2g/L EDTA mixed digested at 8.0X 103The cells were inoculated in subculture flasks (T25) at a cell density of/cm 2 for expansion culture, and the growth and morphological characteristics of the cells were observed under an inverted microscope. The result shows that the second generation of cells have good growth state and uniform shape, and most of the cells are mature mesenchymal stem cells in a wide polygon shape or a flat shape.
3. Construction of matrix-dependent tissue engineering bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells
The construction strategy is shown in figure 1, a Decalcified Bone Matrix (DBM) scaffold material (with the volume of 0.5cm multiplied by 0.3cm) is taken and placed in a 6-well plate, a DMEM culture medium is used for soaking for 48 hours, and the pH value is adjusted to be about 7.2; taking the second generation MSCs in the exponential growth period, washing the MSCs for 2 times by PBS, incubating the MSCs for 1 hour by 0.1% type I collagen (Col I) enzyme, adding 0.25% pancreatin and 0.02% EDTA for digestion for 3-5 min, washing and re-suspending the MSCs by a DMEM medium, mixing and re-suspending the MSCs and endothelial progenitor cells according to the ratio of 1:1, wherein the total number of the planted cells is 107. Cells are inoculated on the decalcified bone matrix scaffold material after the treatment, so that the cell suspension does not overflow out of the scaffold material. After 3 hours the decalcified bone matrix scaffold material was turned over from its bottom to its top surface under aseptic conditions and seeded with the cell suspension as described above. After 3 hours, DMEM medium was added to just over the top surface of the decalcified bone matrix scaffold material, and then 5% CO was added at 37 deg.C2Culturing for 24 hours, and then carrying out osteogenic differentiation induction culture for 13-15 days in vitro, wherein the osteogenic induction culture medium is DMEM (H) + 10% FBS +10mmol/L β -sodium glycerophosphate +0.1 mu mol/L dexamethasone +50mg/LVitC, and the observation of a scanning electron microscope can know that the cells are successfully planted on the scaffold material, then freezing for 48-72 hours at-70-80 ℃, freezing for 24-30 hours, and freezing and storing for 1-3 months so as to obviously reduce immunogenicity, thus forming the matrix-dependent tissue engineering bone based on osteoclast precursors and mesenchymal stem cells as seed cells.
Example 2 in vitro migration, repair and osteogenic differentiation assay of stromal-dependent tissue engineered bone constructed based on mesenchymal stem/endothelial progenitor cells as seed cells
1. In vitro scratch repair experiment: endothelial cells (10)6One/well) were seeded on a 6-well plate and incubated at 37 ℃ for a further period, and when the cells reached 90% growth density, the cell surface was scraped with a 200. mu.l pipette tip, and then the cells were washed with PBS to remove debris, using two protein extracts of a matrix-dependent tissue engineered bone constructed based on mesenchymal stem/endothelial progenitor cells as seed cells (MSC/EPC group) constructed according to the present invention, a matrix-dependent tissue engineered bone constructed based on MSC alone as seed cells (MSC group), and a medium containing 2% serum, respectively, as follows: the culture solution mixed in the proportion of 1 is continuously cultured. The change of the scratched area was observed and observed with a microscope at t0 and 12 hours and the size of the scratched area was measured with Image J. Scratch healing rate (%) (a0-a6)/a0 × 100%, where a0 represents the initial scratch area (t0 hours) and a6 represents the final scratch area at t6 hours. A, B shows scratch repair at the first (t0) for MSC group and MSC/EPC group, respectively, as shown in FIG. 2; C. d is the scratch repair condition after 6 hours (t6) of the MSC group and the MSC/EPC group respectively, the tissue repair ratio of the MSC/EPC group is (61.49 +/-5.85)%, the MSC group is (39.34 +/-3.61)% and the MSC/EPC group is obviously superior to the MSC group of the control (t 5.581, P is 5.581)<0.01)。
2. In vitro migration experiments: cell migration experiments were performed using 24-well plates with bone marrow MSCs at 10 of 500. mu.L6cells/mL are planted in the upper chamber of a Transwell culture system, the lower chamber is a matrix-dependent tissue engineering bone (MSC/EPC group) constructed based on the mesenchymal stem cells/endothelial progenitor cells as seed cells and constructed by the invention, and the matrix-dependent tissue engineering bone (MSC group) with pure MSC as the seed cells is used as a control. After 24 hours cells migrating to the underside of the filter were fixed with 4% paraformaldehyde and stained with DAPI. As shown in fig. 3, DAPI staining for cell migration in the a.msc group; migration of cells in MSC/EPC group DAPI staining. The results showed that the number of cell migration in the MSC/EPC group was (121.63 ± 8.30), the MSC group was (84.97 ± 6.68), and the MSC/EPC group was significantly better than the control MSC group (t ═ 5.96, P<0.01)。
3. In vitro osteogenic differentiation experiments: taking 2 nd generation bone marrow MSC to reach 80% -90% growth density, trypsinizing for 5min, centrifuging at 1000r/min for 3min, preparing cell suspension with F12 culture medium, and culturing at 2 × 105The two protein leaching solutions of matrix-dependent tissue engineering bone (MSC/EPC group) constructed based on the mesenchymal stem cells/endothelial progenitor cells as seed cells, matrix-dependent tissue engineering bone (MSC group) with simple MSC as seed cells and culture medium containing 10% serum, which are respectively inoculated in a 24-well plate, are mixed according to the ratio of 1: the culture solution mixed in proportion of 1 is continuously cultured, alizarin red staining and ALP staining are carried out after 21 days, and the formation condition of calcium nodules is observed by an inverted microscope and is roughly photographed. The results are shown in FIG. 4, wherein A, B in FIG. 4 shows alizarin red staining for MSC group and MSC/EPC group, respectively; c, D of FIG. 4 shows ALP staining for MSC and MSC/EPC groups, respectively. Results show that the results of the two methods of alizarin red staining and ALP staining for evaluating osteogenic differentiation capacity of two matrix-dependent tissue engineering bones are basically consistent, the mineralization proportion of an MSC group is about 38%, and the mineralization proportion of an MSC/EPC group is 45% -50%, so that obvious differences exist.
4. In vitro lumen formation experiment: taking the 2 nd generation EPCs at 5X 104The cells/well are inoculated in a 24-well plate paved with 200 uL/well matrix glue, two protein leaching solutions of matrix-dependent tissue engineering bone (MSC/EPC group) constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells, matrix-dependent tissue engineering bone (MSC group) with simple MSC as seed cells and a culture medium containing 10% serum are respectively used according to the weight ratio of 1: the culture solution mixed at the ratio of 1 was cultured for another 6 hours, and the tube cavity formation was observed using an inverted microscope and 5 fields were randomly selected for photographing. The results are shown in fig. 5, where fig. 5A shows the lumen formation of the MSC group and fig. 5B shows the lumen formation of the MSC/EPC group, and as can be seen from fig. 5, the lumen formation length of the MSC/EPC group is (11.31 ± 0.59) mm, the MSC group is (5.90 ± 0.36)% mm, and the MSC/EPC group is significantly better than the control MSC/EPC group (t ═ 13.56, P<0.001)。
Example 3 repair of bone defects in stromal-dependent tissue engineered bone constructed based on mesenchymal Stem cells/endothelial progenitor cells as seed cells
Animal model preparation and experimental grouping: healthy 2-month-old SD rats, with a body mass (200 + -20) g, were anesthetized and bilateral femoral condyles were exposed under sterile conditions, and bone defects of 3mm in diameter were made with a dental drill. Rats received the following 3 groups of implants to repair bone defects: (1) blank control group: performing debridement treatment only at the defect; (2) control group: matrix-dependent tissue-engineered bone with only MSC as seed cells; (3) experimental groups: the invention relates to a matrix-dependent tissue engineering bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells. Repeatedly administering hydrogen peroxide, iodophor and normal saline to wash defect part, sewing, breeding in cages after the experimental animal revives, and injecting penicillin-streptomycin to prevent infection. Bone defect repair was assessed 2 months after surgery by Micro-CT based on bone volume fraction (BV/TV), trabecular thickness (tb.th), trabecular number (tb.n) and trabecular resolution (tb.sp). And histological observations were performed by Masson staining to evaluate new bone formation at the defect. The results of Micro-CT examination and Masson staining detection after 2 months of planting are shown in FIG. 6, A-C in FIG. 6 are Micro-CT images of 2 months after femoral defect surgery of a blank control group, a control group and an experimental group respectively; D-F in FIG. 6 are Masson staining histological images of femoral defects of blank control group, control group and experimental group for 2 months after surgery, respectively. The result shows that after 2 months of femoral defect operation of a rat, the blank control has almost no new bone formation, a little new bone formation exists in the control group, but no complete bone structure appears at the defect position; the defect repair of the experimental group is basically finished, and the trabecular structure is basically complete. These results indicate that MSC/EPC group matrix-dependent tissue engineered bone has advantages in promoting osteogenesis. Micro-CT examination analysis of fig. 6 a-C showed that the experimental group was significantly stronger in bone volume fraction (BV/TV), number of trabeculae (tb.n) and trabecular thickness (tb.th) than the other two groups (P < 0.01). Masson staining of D-F in FIG. 6 shows a large amount of new bone tissue, intact trabecular bone structure, and more osteoblasts and chondrocytes in part. The control group had a small number of lamellar trabecular bone structures and a small number of osteoblasts. The blank control group is mostly fibrous tissue structure, and no obvious new bone is seen.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (7)
1. The preparation method of the matrix-dependent tissue engineering bone constructed by taking the mesenchymal stem cells/endothelial progenitor cells as seed cells is characterized by planting the mesenchymal stem cells and the endothelial progenitor cells on a porous bone scaffold material according to the proportion of 1:1 to construct a tissue engineering complex, adding an osteogenesis induction culture solution into the tissue engineering complex in vitro to continue culturing for 13-15 days, freezing the tissue engineering complex at the temperature of-70-80 ℃ for 48-72 hours, freeze-drying the tissue engineering complex for 24-30 hours, and freezing and storing the tissue engineering complex for 1-3 months to obtain the matrix-dependent tissue engineering bone.
2. The method for preparing the stromal-dependent tissue engineering bone constructed based on the mesenchymal stem/endothelial progenitor cells as the seed cells according to claim 1, wherein the mesenchymal stem cells are bone marrow mesenchymal stem cells, peripheral blood mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, adipose mesenchymal stem cells or umbilical cord mesenchymal stem cells.
3. The method for preparing the matrix-dependent tissue engineering bone constructed based on the mesenchymal stem/endothelial progenitor cells as the seed cells according to claim 2, wherein the mesenchymal stem cells are umbilical cord mesenchymal stem cells.
4. The method for preparing the matrix-dependent tissue engineering bone constructed based on the mesenchymal stem/endothelial progenitor cells as the seed cells according to claim 1, wherein the porous bone scaffold material is a synthetic bone scaffold material, an acellular bone matrix or a decalcified bone matrix.
5. The method for preparing the matrix-dependent tissue engineering bone constructed based on the mesenchymal stem/endothelial progenitor cells as the seed cells according to claim 4, wherein the porous bone scaffold material is a decalcified bone matrix.
6. The method for preparing the matrix-dependent tissue engineering bone constructed based on the mesenchymal stem cells/endothelial progenitor cells as seed cells according to any one of claims 1 to 5, wherein the decalcified bone matrix scaffold material is taken and soaked in a culture medium for 48 hours, and the pH value is adjusted to 7.2; taking the second-generation mesenchymal stem cells in the exponential growth period, washing the second-generation mesenchymal stem cells by PBS, incubating the second-generation mesenchymal stem cells for 1 hour by using a 0.1 wt% type I collagenase solution, adding pancreatin and a 0.02 wt% EDTA solution for digestion for 3-5 minutes, washing and re-suspending the second-generation mesenchymal stem cells by using a DMEM medium containing 10 wt% fetal calf serum, and mixing and re-suspending the second-generation mesenchymal stem cells and endothelial progenitor cells according to a ratio of 1: 1; inoculating cells to the decalcified bone matrix scaffold material after the treatment, so that the cell suspension just infiltrates the scaffold material and does not overflow out of the scaffold material; turning over the bottom surface of the decalcified bone matrix scaffold material to become a top surface after 3 hours under the aseptic operation condition, and inoculating cell suspension by the same method as the method; the cell scaffold has a regular size of 0.5cm × 0.5cm × 0.3cm, and the total number of planted cells is about 106(ii) a After 3 hours, DMEM medium was added to just over the top surface of the decalcified bone matrix scaffold material, and then 5% CO was added at 37 deg.C2Culturing under the condition; after 24 hours, carrying out osteogenic differentiation induction culture on the cells in vitro for 13-15 days; freezing for 48-72 hours at-70-80 ℃, freeze-drying for 24-30 hours, and freezing and storing for 1-3 months to obviously reduce immunogenicity, so as to form the matrix-dependent tissue engineering bone constructed by taking the mesenchymal stem cells/endothelial progenitor cells as seed cells.
7. The matrix-dependent tissue engineering bone constructed by adopting the preparation method of any one of claims 1 to 6 and based on the mesenchymal stem cells/endothelial progenitor cells as seed cells.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010091980.6A CN111282020A (en) | 2020-02-14 | 2020-02-14 | Matrix-dependent tissue engineering bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010091980.6A CN111282020A (en) | 2020-02-14 | 2020-02-14 | Matrix-dependent tissue engineering bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111282020A true CN111282020A (en) | 2020-06-16 |
Family
ID=71021375
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010091980.6A Pending CN111282020A (en) | 2020-02-14 | 2020-02-14 | Matrix-dependent tissue engineering bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111282020A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113332495A (en) * | 2021-05-31 | 2021-09-03 | 浙江大学 | Three-dimensional vascularized tissue engineering bone and preparation method thereof |
| CN113812398A (en) * | 2021-10-11 | 2021-12-21 | 章毅 | Cryogenic freezing method of scaffolds for tissue repair |
| CN115400149A (en) * | 2022-09-09 | 2022-11-29 | 南京艾尔普再生医学科技有限公司 | A preparation containing MSC cells |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104096266A (en) * | 2014-07-25 | 2014-10-15 | 中国人民解放军第三军医大学 | Tissue-engineered bone based on entochondrostosis system and construction method thereof |
-
2020
- 2020-02-14 CN CN202010091980.6A patent/CN111282020A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104096266A (en) * | 2014-07-25 | 2014-10-15 | 中国人民解放军第三军医大学 | Tissue-engineered bone based on entochondrostosis system and construction method thereof |
Non-Patent Citations (3)
| Title |
|---|
| HADAR ZIGDON GILADI ET AL: "cotransplantation of endothelial progenitor cells and mesenchymal stem cells promote neovascularization and bone regeneration", 《CLINICAL IMPLANT DENTISTRY AND RELATED RESEARCH》 * |
| MOYUAN DENG ET AL: "sustained release of bioactive protein from a lyophilized tissue engineered comstruct promotes the osteogenic potential of mesenchymal stem cells", 《JOURNAL OF ORTHOPAEDIC RESEARCH》 * |
| YUNQING KANG ET AL: "creation of bony microenvironment with CaP and cell derived ECM to enhance human bone marrow MSC behavior and delivery of BMP2", 《BIOMATERIALS》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113332495A (en) * | 2021-05-31 | 2021-09-03 | 浙江大学 | Three-dimensional vascularized tissue engineering bone and preparation method thereof |
| CN113812398A (en) * | 2021-10-11 | 2021-12-21 | 章毅 | Cryogenic freezing method of scaffolds for tissue repair |
| CN113812398B (en) * | 2021-10-11 | 2022-07-29 | 章毅 | Cryogenic freezing method of scaffolds for tissue repair |
| CN115400149A (en) * | 2022-09-09 | 2022-11-29 | 南京艾尔普再生医学科技有限公司 | A preparation containing MSC cells |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108525021B (en) | Tissue engineering skin containing blood vessels and hair follicle structures based on 3D printing and preparation method thereof | |
| Tian et al. | Myogenic differentiation of human bone marrow mesenchymal stem cells on a 3D nano fibrous scaffold for bladder tissue engineering | |
| Yorukoglu et al. | A concise review on the use of mesenchymal stem cells in cell sheet‐based tissue engineering with special emphasis on bone tissue regeneration | |
| Wu et al. | Fibrin glue as the cell-delivery vehicle for mesenchymal stromal cells in regenerative medicine | |
| US9199002B2 (en) | Hybrid soft tissue implants from progenitor cells and biomaterials | |
| JP3808900B2 (en) | Biological material composed of a three-dimensional biocompatible and biodegradable matrix comprising an effective culture of bone marrow stem cells partially or fully differentiated into connective tissue cells and a hyaluronic acid derivative | |
| CN1973910B (en) | Tissue engineering bone | |
| Zhang et al. | Construction of vascularized tissue-engineered bone with a double-cell sheet complex | |
| Gao et al. | Vitalisation of tubular coral scaffolds with cell sheets for regeneration of long bones: a preliminary study in nude mice | |
| Sordi et al. | Three-dimensional bioactive hydrogel-based scaffolds for bone regeneration in implant dentistry | |
| CN111282020A (en) | Matrix-dependent tissue engineering bone constructed based on mesenchymal stem cells/endothelial progenitor cells as seed cells and preparation method thereof | |
| CN101366977B (en) | Tissue mending material with biological activity and preparation method thereof | |
| CN107338218A (en) | Derivant and method of a kind of induced lipolysis stem cell to Chondrocyte Differentiation | |
| CN110478528B (en) | Preparation method and application of novel tissue repair promoting material | |
| CN107137763B (en) | Vascularized tissue engineering bone and preparation method thereof | |
| CN107050517B (en) | Vascularized tissue engineering bone without exogenous support and its prepn | |
| Xu et al. | Fabrication of vascularized and scaffold-free bone tissue using endothelial and osteogenic cells differentiated from bone marrow derived mesenchymal stem cells | |
| CN103223194A (en) | Cartilage graft for cartilage injury repair and preparation method thereof | |
| Fu et al. | Application of 3D-printed tissue-engineered skin substitute using innovative biomaterial loaded with human adipose-derived stem cells in wound healing | |
| CN104250655A (en) | BMP(bone morphogenetic protein)-2/VEGF(vascular endothelial growth factor)165 double gene modified bone marrow mesenchymal stem cells and preparation method thereof | |
| CN101564555A (en) | Tissue engineering bone implant and method for constructing the same | |
| CN112755250A (en) | Tissue engineering peripheral nerve tissue and preparation method thereof | |
| CN114870088B (en) | Preparation method and application of Wnt signal activated bone cell acellular matrix and bone repair stent lined by Wnt signal activated bone cell acellular matrix | |
| US20240091407A1 (en) | Cartilage tissue engineering complex and use thereof | |
| CN111214707A (en) | Matrix-dependent tissue engineering bone with osteoclast precursor and mesenchymal stem cell as seed cells and construction method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200616 |
|
| RJ01 | Rejection of invention patent application after publication |