CN103272288B - Preparation method and application thereof for cell-biological bracket compound based on biological print technology - Google Patents
Preparation method and application thereof for cell-biological bracket compound based on biological print technology Download PDFInfo
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Abstract
The invention provides a preparation method and the application thereof for a cell-biological bracket compound based on biological print technology. The cell-biological compound is formed by fibrous protein which is drawn out from the autoblood of a patient and processed by an ink-jet print technology. The surface and the interior of the cell-biological compound encompass one or more trophic factors and bone mesenchymal stem cells derived from self. According to the invention, the biological print technology is adopted, the appearance shape, cells and encompassing state model of the trophic factors can be designed according to requirements and practical situation, the cell-biological bracket compound is obtained through printing accurately, the fibrous protein and the bone mesenchymal stem cells are derived from self, so that the problem of immunological rejection is avoided, the trophic factors are cultivated in the bracket and are released slowly with the degradation of the bracket material, the cell-biological compound is applied to the spinal cord injured part to promote nerve regeneration and functional reconstruction on the injured part.
Description
Technical field
The present invention relates to a kind of structure of the cell-biological support complex that is used for the treatment of spinal cord injury, especially a kind of preparation method and application thereof of the cell-biological support complex based on biometric print technology.
Background technology
Spinal cord injury is a kind of serious central nervous system trauma disease, has the features such as height expends, high disability rate, brings huge damage and white elephant to society.Because the successional interruption of spinal cord, neuron loss, neurotrophic factor shortage, glial scar and cavity such as form at the difficulty that causes neural regeneration after spinal cord injury.The traditional therapy such as medicine, operation cannot solve the problem of regeneration, and the immunologic rejection that acellular support source is limited and existence is larger.Basic research is in recent years the development of neural tissue engineering technology particularly, after spinal cord injury, aspect the research of neurological functional recovery, has obtained compared with much progress, makes neuranagenesis and reparation become possibility.
Neural tissue engineering technology is to implant kind of a three-dimensional biological engineering material that is implanted with cell at damage location, to reach the object of repairing injured nerve tissue.Tissue engineering material mainly contains natural macromolecular material and degradable macromolecule synthetic material.Natural macromolecular material has good biocompatibility, can promote in vitro adhesion, propagation and the differentiation of cell, and convenient carrying neurotrophic factor, good with the conformability of myeloid tissue, can reduce inflammatory reaction and reduce the cicatrix that proliferation of astrocytes forms, can promote to a certain extent the recovery of impaired spinal cord structure and function.Natural macromolecular material comprises collagen, chitosan and alginate etc.Wherein fibrin can obtain from blood samples of patients, do hydrogel support with patient's self fibrin, alleviate the biomimetic scaffolds immunologic rejection producing that implants, and in fibrin hydrogel, contain transforming growth factor β and platelet derived growth factor, can promote that migration, propagation and the extracellular matrix of cell are synthetic, thereby have broad application prospects.
Seed cell for repair of spinal cord injury mainly contains stem cell, Olfactory essheathing cell, schwann cell etc. at present.Mesenchymal stem cells MSCs (bone marrow mesenchymal stem cells, MSCs) is the non-hematopoietic stem cell of hematopoietic stem cells in bone marrow, and it in vivo, external all to have across differentiation of germinal layers be the potential of neurocyte and glial cell.Research shows, Bone Marrow Mesenchymal Stem Cells Transplantation can move to damage location in spinal cord injury model, and is divided into neuron and neurogliocyte under the effect of local microenvironment, substitutes damaging cells, neural circuit reconstruction, and secretory nerve trophic factors promotes the reparation of spinal cord injury.MSCs draws materials conveniently, amplification is rapid, can realize autotransplantation, avoids immunologic rejection and ethics constraint, is a kind of seed cell of current extensive use.
Neurotrophic factor (neurotrophic factors, NTFs) is a class has the effect of maintaining peptide growth factor to neuronal survival, growth, and in injury region microenvironment, the shortage of NTFs may be one of key factor of neuranagenesis difficulty.It is the neuronic plasticity of scalable and promotion neuranagenesis in the time of spinal cord injury, therefore applying ectogenic NTFs treatment spinal cord injury is one of current study hotspot, applies more have nerve growth factor (NGF), Brain Derived Neurotrophic Factor (BDNF) and neurenergen 3 (NT-3) etc.Research shows, BMSCs transplantation treatment spinal cord injury and neurotrophic factor have synergism as BDNF, NGF etc.
Research shows, therapeutic alliance strategy is expected to make repair of spinal cord injury to reach better effect, as neurotrophic factor and cell-biological support complex use in conjunction.But traditional Method of Tissue Engineering is to be based upon the solid support of making in advance with on the patient's normal tissue cell basis separating, after being cultivated to amplification, patient's normal tissue cell is inoculated in the degradable biological support of manufacturing in advance, cell-biological support complex is transplanted in patient body after cultivating in vitro, degradable biological support is constantly degraded until disappear, cell proliferation and differentiation in support simultaneously, finally forms the morphology and function substitute consistent with normal structure and organ in vivo.Although this method has obtained some immense successes at structure cavity tissue and organ simple in structure, but there are many deficiencies in this method, particularly in the time building the tissue of internal structure complexity and organ, in this method, cell cannot precision planting to internal stent, it is random absorption surface, cell active factor can only affect the Growth and Differentiation of rack surface cell, but the control of internal stent cytoactive is difficult to accomplish, in view of the limitation of traditional Method of Tissue Engineering, the tissue engineering technique of finding science is more extremely urgent.
Inkjet technology is the untouchable printing technique that ink droplet is ejected into acceptor formation image or word, and it is not only applied to office ink-jet printer, and is used successfully in medical science and biomedical engineering.The scraps of paper of biometric print are designed to the one degradable biological scraps of paper in vivo in theory; The ink of biometric print is designed in theory the cell solution of speciality or has bioactive trophic factors solution.This special solution is ejected on the degradable biological scraps of paper, again that the scraps of paper are stacking in certain sequence after printing.Use biometric print technology, cell/trophic factors can be attached to predetermined position accurately.Along with the research of this technology deepens continuously, research shows that inkjet technology is applicable to print cell, biologic bracket material and cell active factor, its application in Organ printing also receives publicity day by day, and this is that organizational project has been brought new hope in the successful utilization building in bionical tissue and organ.
China Patent Publication No. CN101829361A discloses a kind of nano-bionic material for tissue repair and preparation method thereof, described nano-bionic material comprises nano bionic support and the hydrosol attached to it, is coated with one or more trophic factors/cells in the hydrosol.Described in it, preparation method comprises preparation electrospinning solution and the aqueous solution that contains trophic factors/cell; Make nano bionic support with electrostatic spinning; The aqueous solution that contains trophic factors/cell is printed to the first-class step of nano bionic support with ink-jet printer.
China Patent Publication No. CN102908207A provides a kind of tissue engineering nerve graft based on biometric print technology and preparation method thereof, and described tissue engineering nerve graft comprises fibrous framework in outer tube and pipe, and surfaces externally and internally can be coated trophic factors/cell.Utilize biometric print technology, carry out high-true emulation printing according to neural actual form demand, size, quantity, nozzle by adjusting inkjet printing machine nozzle is to distance, the supercharger pulse frequency of bottom and work out the control sequence of specific printing, polymer material solution is printed as to specific nerve graft with ink-jet printer.
In the recent period, there is researcher to use inkjet technology to print layer by layer fibrin monomer and thrombin, successfully form 3D fibrin hydrogel support, scanning electron microscopic observation 3D fibrin scaffold which, show by the porous of fibrin hydrogel and the morphological analysis of fibrin fiber, fibrin hydrogel support has and presents a loose porous micro structure, and this structure can more effectively be cell delivery nutrition and the oxygen in support.
To sum up, patent of the present invention is by extracting the fibrin in patient's self-blood, utilize biometric print technology to print one deck fibrin hydrogel layer, then on hydrogel layer, print neurotrophic factor and the BMSCs that derives from patient self, as above step is layering, thereby is formed for cell-biological support complex that spinal cord injury is transplanted.
Summary of the invention
The object of the present invention is to provide a kind of preparation method and application for the treatment of spinal cord injury thereof of the cell-biological support complex based on biometric print technology, to promote neuranagenesis and the function reparation of damage location.
The preparation method of the cell-biological support complex based on biometric print technology that the present invention proposes, obtains patient's self BMSCs and fibrin, the aqueous solution that preparation contains trophic factors/cell; Utilize biometric print technology to print one deck fibrin monomer and thrombin, obtain fibrin hydrogel layer; On hydrogel layer, print neurotrophic factor and BMSCs; Be layering, thereby be formed for cell-biological support complex that spinal cord injury is transplanted; Concrete steps are as follows:
(1). in autoblood, extract Fibrinogen, extract experimenter's blood, prepare Fibrinogen;
(2). prepare autologous bone marrow mesenchymal stem cells (BMSCs) solution;
(3). prepare neurotrophic factor aqueous solution, described neurotrophic factor be one in basic fibroblast growth factor (bFGF), brain derived neurotrophic factor (BDNF), NT3 (NT-3) or glial cell line-derived neurotrophic (GDNF) to several, the amount of neurotrophic factor on cell-biological support complex is 1 μ g/g-10mg/g.
(4). the printing of cell-biological support complex
(4.1) Fibrinogen step (1) being obtained is dissolved in PBS, is configured to the solution that concentration is 10mg/mL, is loaded in ink-cases of printers; Thrombin is dissolved in GaCl
2solution, is configured to the solution that concentration is 20IU/mL, is loaded in another print cartridge;
(4.2) adjusting with the printer nozzle syringe needle diameter that Fibrinogen print cartridge is housed is 200 μ m, and syringe needle quantity is 8, and nozzle is 50mm to the distance of bottom, supercharger pulse frequency 15v;
Adjusting with the printer nozzle syringe needle diameter that thrombin print cartridge is housed is 100 μ m, and syringe needle quantity is 10, and nozzle is 25mm to the distance of bottom, supercharger pulse frequency 15v;
(4.3) according to the actual requirements, the shape of design cell-biological support complex is printed skim Fibrinogen on coverslip, changes the shower nozzle that thrombin print cartridge is housed, and prints three times continuously gel time 3-5min on Fibrinogen thin layer.
(4.4) adjusting with the printer nozzle syringe needle diameter that autologous bone marrow mesenchymal stem cells print cartridge is housed is 100 μ m, and syringe needle quantity is 6, and nozzle is 25mm to the distance of bottom, supercharger pulse frequency 15v;
Adjusting with the printer nozzle syringe needle diameter that one or more neurotrophic factor print cartridges are housed is 50-200 μ m, and syringe needle quantity is 3-6, and nozzle is 25mm to the distance of bottom, supercharger pulse frequency 15v;
(4.5) according to the actual requirements, the coated form of design cell and trophic factors, works out corresponding control sequence, on above-mentioned fibrin hydrogel, prints autologous bone marrow mesenchymal stem cells and neurotrophic factor.
Repeating step (4.1)-(4.5), until obtain order target cell-biological support complex, the needs of the realistic transplanting of its shape, rack body is fibrin hydrogel, and rack surface and inside are coated with autologous bone marrow mesenchymal stem cells and one or more neurotrophic factors.
In the present invention, the printer 55C of the Hewlett-Packard ink-jet printer that described ink-jet printer is preferably reequiped, adopting print cartridge model is HP51626A.
Utilize the application in treatment spinal cord injury of the cell-biological support complex based on biometric print technology that the inventive method prepares.
Compared with prior art, the present invention has remarkable advantage:
(1) the present invention utilizes biometric print technology, can design a model with practical situation as required, and precise Printing obtains the transplanting of cell-biological support complex for spinal cord injury.
(2) the biometric print technology that the present invention uses is simple, and cost is lower, and production process safety is controlled.
(3) the present invention's fibrin used derives from patient self, without all multi-risk Systems such as immunologic rejection, disease propagation, and degraded automatically after transplanting, catabolite is absorbed completely by human body, has avoided immuning tissue's reaction.
(4) the present invention's seed cell used is mesenchymal stem cells MSCs, and its source is abundant, draw materials conveniently, easily separation and purification, and derive from autologous, without immunologic rejection.
(5) seed cell of the present invention and neurotrophic factor are planted in internal stent, slowly discharge long term with the degraded of timbering material.
Detailed description of the invention
further illustrate the present invention below by embodiment.
embodiment 1:
1. in autoblood, extract Fibrinogen
Extract the about 200mL of experimenter's blood, Fibrinogen preparation method is referring to patent publication No. CN102286095A.
2. the preparation of autologous bone marrow mesenchymal stem cells (BMSCs) solution
Extract the about 40mL of experimenter's bone marrow, anticoagulant heparin, note avoiding grumeleuse to form as far as possible, bone marrow is transferred in super-clean bench in 2 aseptic centrifuge tubes of 50mL, respectively by PBS proportional diluted and mix, draw FICOLL10-20mL/ pipe × 4 pipes, inclination centrifuge tube, bone marrow suspension is slowly added to FICOLL surface along wall respectively, make it to form separating surface clearly, 22 DEG C, 1350 revs/min, centrifugal 30 minutes, steadily take out centrifuge tube, in super-clean bench, middle tunica albuginea layer is taken out, be placed in aseptic centrifuge tube, add PBS to 40mL to mix, 1500 revs/min, centrifugal 5 minutes, abandon supernatant, add MESENC μ LT culture fluid 10mL, piping and druming evenly, obtained cell suspension 20 μ l are in EP pipe, add again the 180 μ l dilutions of leukocyte diluent, mix in agitator, getting 20 μ l is added on cell counting count board, counting: the mean × 10-E of four-quadrant total cellular score
6for total cellular score.Press 2-5 × 10-E
6/ ware seeds cells in culture dish, and adds fresh medium, makes final volume reach 7-8mL, puts into incubator.Within every 3 days, change liquid 1 time, go down to posterity until Growth of Cells reaches the 70-80% of culture dish bottom later.Within cell 3-4 after going down to posterity days, half amount is changed liquid 1 time, and Growth of Cells area reaches culture dish bottom 80-90% and need go down to posterity again.In passage process, directly collect BMSCs or collection thaw after through the MSCs of washing, add that to make cell suspension containing 20% albuminous normal saline for subsequent use, making cell density is 1 × 10
6-1 × 10
8/ mL.
BMSCs carries out flow cytometer identification of cell type for before auxiliary treatment; Chromosome detects gets rid of diploid variation; The nosetiology that moves ahead infusion detects; Cell suspension skin test.
3. the preparation of neurotrophic factor aqueous solution
Described neurotrophic factor is one or more in basic fibroblast growth factor (bFGF), brain derived neurotrophic factor (BDNF), NT3 (NT-3), glial cell line-derived neurotrophic (GDNF), and the amount of neurotrophic factor on cell-biological support complex is 1 μ g/g-10mg/g.
4. the printing of cell-biological support complex
4.1 Fibrinogens are dissolved in PBS, and the solution that configuration final concentration is 10mg/mL, is loaded in the print cartridge that model is HP51626A; Thrombin is dissolved in GaCl
2solution, configuration final concentration is the solution of 20I μ/mL, is loaded in another print cartridge.
The 4.2 printer nozzle syringe needle diameters of adjusting and be equipped with Fibrinogen print cartridge are 200 μ m, and syringe needle quantity is 8, and nozzle is 50mm to the distance of bottom, supercharger pulse frequency 15v;
Adjusting with the printer nozzle syringe needle diameter that thrombin print cartridge is housed is 100 μ m, and syringe needle quantity is 10, and nozzle is 25mm to the distance of bottom, supercharger pulse frequency 15v;
Many tubular structures that the macroscopic view of 4.3 design cell-biological support complex is configured as being arranged in parallel, relevant parameter is set on computers and on coverslip, prints skim Fibrinogen, the shower nozzle of thrombin print cartridge is equipped with in replacing, on Fibrinogen thin layer, print three times continuously gel time 3-5min.
The 4.4 printer nozzle syringe needle diameters of adjusting and be equipped with autologous bone marrow mesenchymal stem cells print cartridge are 100 μ m, and syringe needle quantity is 6, and nozzle is 25mm to the distance of bottom, supercharger pulse frequency 15v;
Adjusting with the printer nozzle syringe needle diameter that one or more neurotrophic factor print cartridges are housed is 50-200 μ m, and syringe needle quantity is 3-6, and nozzle is 25mm to the distance of bottom, supercharger pulse frequency 15v;
4.5 according to the actual requirements, and the coated form of design cell and trophic factors, works out corresponding control sequence, on above-mentioned fibrin hydrogel, prints autologous bone marrow mesenchymal stem cells and neurotrophic factor.
Repeating step 4.1-4.5, until obtain order target cell-biological support complex, many tubular structures that its macroscopic view is configured as being arranged in parallel, rack body is fibrin hydrogel, rack surface and inside are coated with autologous bone marrow mesenchymal stem cells (cell density 1 × 10
6/ mL) and one or more neurotrophic factors (amount is 1 μ g/g-10mg/g).
Claims (2)
1. a preparation method for the cell-biological support complex based on biometric print technology, is characterized in that adopting patient's self BMSCs and Fibrinogen; The aqueous solution that preparation contains trophic factors/cell; Utilize biometric print technology to print one deck Fibrinogen, and print thrombin thereon, obtain fibrin hydrogel layer; On hydrogel layer, print neurotrophic factor and BMSCs; Be layering, thereby be formed for cell-biological support complex that spinal cord injury is transplanted; Concrete steps are as follows:
(1). in autoblood, extract Fibrinogen;
(2). prepare autologous bone marrow mesenchymal stem cells solution;
(3). prepare neurotrophic factor aqueous solution, described neurotrophic factor be one in basic fibroblast growth factor, brain derived neurotrophic factor, NT3 or glial cell line-derived neurotrophic to several, the amount of neurotrophic factor on cell-biological support complex is 1 μ g/g-10mg/g;
(4). the printing of cell-biological support complex
(4.1) Fibrinogen step (1) being obtained is dissolved in PBS, is configured to the solution that concentration is 10mg/mL, is loaded in ink-cases of printers; Thrombin is dissolved in CaCl
2solution, is configured to the solution that concentration is 20IU/mL, is loaded in another print cartridge;
(4.2) adjusting with the printer nozzle syringe needle diameter that Fibrinogen print cartridge is housed is 200 μ m, and syringe needle quantity is 8, and nozzle is 50mm to the distance of bottom, supercharger pulse frequency 15v;
Adjusting with the printer nozzle syringe needle diameter that thrombin print cartridge is housed is 100 μ m, and syringe needle quantity is 10, and nozzle is 25mm to the distance of bottom, supercharger pulse frequency 15v;
(4.3) according to the actual requirements, the shape of design cell-biological support complex is printed skim Fibrinogen on coverslip, changes the shower nozzle that thrombin print cartridge is housed, and prints three times continuously gel time 3-5min on Fibrinogen thin layer;
(4.4) adjusting with the printer nozzle syringe needle diameter that autologous bone marrow mesenchymal stem cells print cartridge is housed is 100 μ m, and syringe needle quantity is 6, and nozzle is 25mm to the distance of bottom, supercharger pulse frequency 15v;
Adjusting with the printer nozzle syringe needle diameter that one or more neurotrophic factor print cartridges are housed is 50-200 μ m, and syringe needle quantity is 3-6, and nozzle is 25mm to the distance of bottom, supercharger pulse frequency 15v;
(4.5) according to the actual requirements, the coated form of design cell and trophic factors, works out corresponding control sequence, on above-mentioned fibrin hydrogel, prints autologous bone marrow mesenchymal stem cells and neurotrophic factor;
Repeating step (4.1)-(4.5), until obtain order target cell-biological support complex, the needs of the realistic transplanting of its shape, rack body is fibrin hydrogel, and rack surface and inside are coated with autologous bone marrow mesenchymal stem cells and one or more neurotrophic factors.
2. method according to claim 1, is characterized in that the printer 55C of the Hewlett-Packard ink-jet printer that described ink-jet printer is preferably reequiped, and adopting print cartridge model is HP51626A.
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| CN105030386A (en) * | 2015-07-09 | 2015-11-11 | 中国人民武装警察部队后勤学院附属医院 | Spinal cord micro-catheter stent fast to form and manufacturing method thereof |
| CN107418998B (en) * | 2016-09-14 | 2020-02-28 | 四川蓝光英诺生物科技股份有限公司 | Microparticle manipulation method, bioprinting method and biological construct |
| CN107432955B (en) * | 2016-09-14 | 2020-09-04 | 四川蓝光英诺生物科技股份有限公司 | Methods and kits for making biological constructs |
| US11439731B2 (en) | 2016-09-14 | 2022-09-13 | Revotek Co., Ltd. | Artificial tissue progenitor and method for preparing the same |
| US20210038762A1 (en) * | 2018-01-31 | 2021-02-11 | Rokit Healthcare Inc. | Bioink composition for cartilage regeneration, method for manufacturing customized scaffold for cartilage regeneration using same, and customized scaffold for cartilage regeneration manufactured using manufacturing method |
| MX2020008034A (en) * | 2018-01-31 | 2020-09-17 | Rokit Healthcare Inc | Bioink composition for dermis regeneration sheet, method for manufacturing customized dermis regeneration sheet using same, and customized dermis regeneration sheet manufactured using manufacturing method. |
| EP3939625A4 (en) * | 2019-03-11 | 2022-12-07 | Rokit Healthcare Inc. | Method for producing composition for regenerating cartilage using freeze-dried hyaline cartilage powder, composition for regenerating cartilage produced using same, method for producing scaffold for regenerating patient-specific cartilage using composition for regenerating cartilage, and scaffold for regenerating patient-specific cartilage |
| WO2022073090A1 (en) | 2020-10-06 | 2022-04-14 | Janaina De Andrea Dernowsek Ltda | Method for producing an extracellular matrix protein composition and product obtained by said method |
| CN114366383B (en) * | 2021-06-11 | 2023-01-17 | 冯世庆 | Bionic spinal cord stent for promoting axon directional extension after spinal cord injury |
| CN113679885A (en) * | 2021-08-19 | 2021-11-23 | 中国人民解放军联勤保障部队第九八八医院 | Method for repairing and reconstructing craniomaxillofacial surface based on 3D printing construction |
| CN114306731B (en) * | 2021-12-28 | 2022-10-14 | 中国科学院上海硅酸盐研究所 | Multi-cell printing active scaffold for neurogenic bone regeneration and preparation method and application thereof |
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