CN113576719A - Bionic microchannel integrated intervertebral disc stent and preparation method and application thereof - Google Patents
Bionic microchannel integrated intervertebral disc stent and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of bionic scaffold preparation, and particularly relates to a bionic microchannel integrated intervertebral disc scaffold, and a preparation method and application thereof, wherein the preparation method comprises the following steps of 1) preparing a fibrous ring tissue bionic microchannel pore-forming template; 2) preparing a pore-forming material with a bionic porous structure of nucleus pulposus tissues; 3) assembling a complete intervertebral disc bionic microchannel pore-forming template; 4) pouring a microchannel solution into the bionic microchannel pore-forming template; 5) and eluting and leaching template materials in the fibroin scaffold. The intervertebral disc scaffold obtained by preparing the micro-channel template and the nucleus pulposus template and then filling the micro-channel solution has two tissue structures of bionic reconstruction of the fibrous ring and the nucleus pulposus, so that the integrated bionic micro-channel intervertebral disc scaffold is formed.
Description
Technical Field
The invention belongs to the field of bionic scaffold preparation, and particularly relates to a bionic microchannel integrated intervertebral disc scaffold as well as a preparation method and application thereof.
Background
Intervertebral disc degeneration is the main reason causing chronic low back pain and neck pain at present, brings great pain to patients themselves and brings great economic burden to families and society. Current treatment methods for intervertebral disc degeneration are mainly based on conservative treatment and surgical treatment: conservative treatment methods mainly include drug therapy and physical therapy (acupuncture, traction, etc.), the intervertebral disc is used as a small cell tissue lacking blood supply, the regeneration capability is poor, and the long-term effect of conservative treatment is usually poor; surgical procedures primarily include discectomy, spinal fusion, and disc replacement, with a high risk of recurrence due to the frequent resulting spinal biomechanical changes and degeneration of the adjacent segment discs as a result of the surgical treatment procedure.
The normal intervertebral disc is mainly composed of three parts, namely a highly hydrated nucleus pulposus, an annulus fibrosus rich in collagen fibers and cartilage endplates at two ends: the normal nucleus pulposus tissue is positioned in the center of the intervertebral disc, the main chemical components are type II collagen and proteoglycan, and the normal nucleus pulposus tissue is in a jelly shape due to rich water content; the fibrous ring tissue is mainly formed by overlapping 15-25 collagen fiber sheets, is annularly wrapped on the periphery of nucleus pulposus, the crossing angle of collagen fiber bundles in adjacent sheets is about 60 degrees, and the main chemical component is type I collagen; the cartilage end plates are positioned at the upper end and the lower end, connect the annulus fibrosus and the nucleus pulposus into a whole, and have the main function of serving as a main place for exchanging nutrient substances and metabolic products of the intervertebral disc. It is due to the complex spatial structure of the intervertebral disc that the disc plays an important role in carrying loads, damping shocks, performing flexion and extension and torsion movements and maintaining intervertebral height. Current clinical strategies for disc degeneration, while effective in relieving pain in patients, do not restore the biological function of the disc. The tissue engineering bionic technology based on the biological material provides great potential for biological repair of intervertebral disc degeneration.
The research and development of biological materials are indispensable factors in the field of tissue engineering, and an important basis is provided for the construction of bionic tissues and bionic organs. At present, the scaffold material in the intervertebral disc tissue engineering mainly comprises two types of natural materials and synthetic materials: the natural materials mainly comprise silk fibroin, collagen, hyaluronic acid, chitosan and the like, and are similar to components of natural extracellular matrix (ECM) and have good biocompatibility and biodegradability, so the material is widely applied to the research of intervertebral disc tissue engineering. Among them, Silk Fibroin (SF) is used as a degradable natural biomaterial approved by FDA, has biocompatibility equivalent to collagen and hyaluronic acid, has outstanding characteristics of low immunogenicity, appropriate mechanical properties, abundant sources and the like, and has been widely used in the field of tissue engineering such as bone, cartilage, intervertebral disc and ligament. The synthetic materials mainly comprise Polycaprolactone (PCL), polyglycolic acid (PGA), polylactic-polyglycolic acid copolymer (PLGA), Polyurethane (PU) and the like, and the materials become common materials of the intervertebral disc tissue engineering scaffold by virtue of the characteristics of excellent mechanical properties and easiness in repeated processing and shaping. Among them, PCL is the most common scaffold material for tissue engineering of annulus fibrosus, because compared with other materials, it has superior physical properties such as stronger elasticity and hardness, and can be processed into tissue engineering scaffolds of various fiber diameters and different fiber arrangement structures by various tissue engineering techniques (such as electrospinning, wet spinning, melt spinning, etc.).
In recent years, with the development of tissue engineering techniques, a number of tissue engineering approaches have been attempted to create tissue engineered disc scaffolds that highly mimic the structure and function of the natural intervertebral disc for the repair of degenerated intervertebral discs. Loss of nucleus pulposus water is considered to be the main pathological manifestation of disc degeneration, and therefore early disc tissue engineering has focused primarily on how to restore nucleus pulposus hydration status. The hydrogel has a gel form similar to that of nucleus pulposus and is rich in water content, so that various hydrogels are developed for research on the regeneration and repair of nucleus pulposus tissues and achieve more satisfactory effects. The complex collagen lamellar arrangement structure in the fibrous ring endows the fibrous ring with the complex mechanical load generated by resisting the spinal motion, and the difficulty of effectively and bionically reconstructing the complex space structure of the fibrous ring is still a problem of the tissue engineering fibrous ring. Electrospinning is one of the common histological engineering methods used to construct biomimetic fibrous ring scaffolds: nerrkar obtains an oriented nano fibrous membrane by using an electrostatic spinning method, then tries to construct a tissue engineering fibrous ring support through lamination assembly, and the result shows that the fiber membrane with reverse cross lamination has stronger mechanical property than the fiber membrane with parallel lamination, and meanwhile, the cell experiment result shows that the oriented nano fibrous membrane can guide mesenchymal stem cells to grow along the parallel fiber orientation and the extracellular matrix to regularly deposit, so that a new idea is provided for constructing the tissue engineering fibrous ring through lamination assembly of the fiber membrane; chu et al prepared 4 kinds of polyether carbonate urea fiber scaffolds with different rigidity and fiber size by electrospinning to study the induced differentiation potential of different scaffold structures on the fiber ring cells, and found that the fiber ring cells inoculated on the surface of the scaffold exhibited different gene expression patterns only due to the different rigidity and fiber size of the scaffold. Although electrospun fibrous membranes are used by many for tissue engineering of the annulus fibrosus, electrospun fibrous membranes often have difficulty in growing tissue and cells into the interior of the scaffold due to the thin diameter of the fibers and the close arrangement of the fibers. Wet spinning is a common tissue engineering method for preparing oriented fiber scaffolds, and because the deposition rate of the spun fibers in the solution is lower than that of air, the method can prepare fiber scaffolds with larger pore structures. The Zhu group research proves that the oriented fiber membrane with the fiber diameter of micron grade can be obtained by using the wet spinning technology, the cell infiltration growth can be better supported, and the Zhu group research attracts important attention in the field of tissue engineering. Although the wet spinning technology provides great help for the development of tissue engineering, the technology is mainly suitable for preparing an oriented fiber scaffold, and the application range of the technology has certain limitation because the winding angle of the spun fiber is difficult to control and the oriented crossed fiber scaffold is difficult to obtain. The freeze drying technology is also a commonly used preparation method of the tissue engineering scaffold at present: the Chang group obtains a porous fiber ring scaffold by freeze drying, and then cultures and observes after inoculating the fiber ring cells to the scaffold, and the porous structure of the fiber ring scaffold can provide a carrier and a growth space for the fiber ring cells. A Mandal research team uses a directional freezing technology to respectively prepare a porous 3D silk fibroin scaffold with a directional fiber structure and a silk fibroin sheet scaffold with directional fibers, and the result proves that the porous scaffold with the directional fiber structure prepared by the method is beneficial to the adhesion and proliferation of porcine anulus cellularis cells, chondrocytes and mesenchymal stem cells and can influence the growth morphology of the cells. The melt spinning technology is also a common spinning technology of a tissue engineering fiber scaffold, the method replaces the high-voltage electric spraying principle in electrostatic spinning with a high-temperature melting principle, does not use any toxic reagent and does not contact high voltage electricity in the operation process, and the method has the characteristics of safe and convenient operation and no damage to human bodies. In addition, the method can obtain fiber scaffolds with different crossing angles or different diameter ranges by adjusting parameters, and is already applied to bionic construction of various tissues such as blood vessels, ligaments and intervertebral discs. Summer and the like prepare the fibrous ring bionic scaffold with uniform fiber thickness and 60-degree directional intersection by adjusting parameters such as the propelling speed, the rotating speed of a receiving rod, the horizontal moving speed and the like and using a melt spinning technology. However, in the later stages of disc degeneration, both the annulus fibrosus and nucleus pulposus tissues exhibit significant pathological degenerative manifestations, including: decreased water in nucleus pulposus tissue, decreased extracellular matrix components (I I type collagen and proteoglycans); the structure of the fibrous ring tissue collagen fiber sheet layer is disordered, and the contents of extracellular matrix degrading enzymes and inflammatory cytokines are increased. Aiming at the seriously degenerated intervertebral disc tissue, the long-term effect of simply repairing the nucleus pulposus tissue or the annulus fibrosus tissue has limitation, so that how to effectively realize the biological repair of the nucleus pulposus tissue and the annulus fibrosus tissue simultaneously has important significance.
In summary, the ideal tissue engineering disc scaffold should have the following characteristics: firstly, the stent material has good biocompatibility and proper biodegradability, and the degradation speed of the stent material should be synchronous with the tissue regeneration process; secondly, the scaffold can bionically reconstruct the complex anatomical structure of the annulus fibrosus and/or the nucleus pulposus in the aspect of structure, and provides a good carrier microenvironment for cell and tissue regeneration; finally, the scaffold should have suitable mechanical properties, which are key factors for achieving intervertebral disc regeneration repair.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a bionic microchannel integrated intervertebral disc stent and a preparation method and application thereof.
In order to achieve the purpose, the invention adopts the technical scheme that:
a preparation method of a bionic microchannel integrated intervertebral disc scaffold comprises the following steps:
1) preparing a bionic microchannel pore-forming template of a fibrous ring tissue: preparing fibers with the inner diameter of 2-8mm and the outer diameter of 4-14mm by using a melt spinning technology; and the fibers are arranged in a 60-degree oriented crossed and laminated way to form a long tubular fiber ring pore-forming scaffold template;
2) preparing a pore-forming material with a bionic porous structure of nucleus pulposus tissue: preparing nucleus pulposus template microspheres by adopting a dispersion solidification method;
3) assembling a complete intervertebral disc bionic microchannel pore-forming template: 3.1) drying and cutting the long tubular fiber ring pore-forming support template obtained in the step 1); 3.2) sleeving a silicone tube on the outer layer of the cut fiber ring pore-forming scaffold template; 3.3) filling the center of the fiber ring pore-forming template bracket sleeved with the silicone tube with the nucleus pulposus template microspheres obtained in the step 2).
4) Injecting a micro-channel solution into the bionic micro-channel pore-forming template: completely soaking the complete bionic microchannel pore-forming template of the intervertebral disc obtained in the step 3) in a microchannel solution, and repeatedly sucking the microchannel solution by using a vacuum drying pump under negative pressure to promote the microchannel solution to be fully filled into the internal gap of the complete pore-forming template; freezing, molding and drying;
5) elution leaching of template material in fibroin scaffolds: 5.1) removing redundant silk fibroin materials at the periphery of the freeze-dried silk fibroin bracket and an outer layer silicone tube to obtain a silk fibroin/fiber bracket/nucleus pulposus template composite bracket; 5.2) soaking the silk fibroin/fiber scaffold/nucleus pulposus template composite scaffold in absolute ethyl alcohol to denature the silk fibroin; 5.3) fully soaking, eluting and leaching the fiber scaffold material in the composite scaffold by using an organic solvent; 5.4) fully washing the bracket by absolute ethyl alcohol to remove residual reagent; 5.5) leaching the nucleus pulposus template material by using n-hexane for elution by a soxhlet extractor; 5.6) removing residual n-hexane by vacuum drying pump negative pressure suction; storing for later use.
The step 1) comprises the following specific steps:
1.1) placing the fiber support in a high-temperature melting reaction furnace of a melt spinning machine, and completely melting the fiber support into a liquid state;
1.2) setting parameters of a control panel of the melt spinning machine as follows: the temperature of the melting reaction furnace is 180-210 ℃, the rotating speed of the receiving rod is 50-150rpm, and the moving speed of the horizontal worktable is 50-100 Hz; adjusting the propelling speed of the micro-injection pump to be 1-5 mL/min; selecting the diameter of the receiving rod to be 2-14mm, adjusting the receiving distance to be 1-3cm, and adjusting the specification of the syringe needle to be 14-20G;
1.3) slowly extruding the molten fiber support material liquid by a metal syringe needle, uniformly drawing the fiber support material in a liquid state, successfully winding the fiber support material on a rotating shaft center with the diameter of 2-14mm, and removing the shaft center to obtain a long tubular fiber ring pore-forming support template with proper inner diameter and outer diameter and fibers in 60-degree oriented crossed laminated arrangement.
Preferably, the parameters of the control panel of the melt spinning machine set in the step 1.2) are as follows: the temperature of the melting reaction furnace is 200 ℃, the rotating speed of a receiving rod is 120rpm, and the moving speed of a horizontal workbench is 60 Hz; adjusting the propelling speed of the micro-injection pump to be 2 mL/min; the diameter of the receiving rod is selected to be 2mm, the receiving distance is adjusted to be 1.5cm, and the specification of the syringe needle is 16G. Thus obtaining the fiber with the preferred inner diameter of 2mm and the outer diameter of 4 mm; the fiber is in 60 degrees, oriented, crossed and laminated arrangement with the long tubular fiber ring pore-forming scaffold template; the nucleus pulposus template microspheres are paraffin microspheres, and the specific method comprises the following steps:
2.1) accurately weighing 60 parts by mass of paraffin blocks, adding the paraffin blocks into a PVA solution containing 1200 parts by volume of 0.5 wt%, placing the mixture on a constant-temperature magnetic stirrer, and continuously stirring the mixture at the temperature of 60 ℃ until the paraffin is completely melted and uniformly dispersed in the PVA solution to form paraffin microspheres;
2.2) continuing to maintain the stirring state after stopping heating, quickly adding ice water, and continuing to stir for a plurality of minutes to solidify and form the emulsified paraffin wax microspheres;
2.3) carrying out size sorting by using sieves with the aperture gradients of 40-60 meshes, 60-80 meshes, 80-100 meshes, 100-140 meshes and 120-140 meshes, fully washing with running water to remove PVA, and drying after alcohol dehydration;
2.4) preparing the paraffin microspheres with different grain diameters by the method, subpackaging and storing in a refrigerator at 4 ℃ for later use.
The specific steps of the step 3) are as follows: 3.1) placing the long tubular fiber ring pore-forming support template prepared by the melt spinning technology in a constant-temperature air-blast drying oven to be heated for 10min at 60 ℃; cutting the product into 1cm length with a blade after cooling; 3.2) sleeving a silicone tube with a proper inner diameter on the outer layer of the long tubular fiber ring pore-forming scaffold template; 3.3) nucleus pulposus template microspheres with the size of 80-120 meshes are filled in the center of the long tubular fiber ring pore-forming stent template; heating at 55 deg.C for 20 min; cooling to room temperature, and completing the preparation of the complete intervertebral disc pore-forming template.
The micro-channel solution in the step 4) is one of silk fibroin solution, chitosan solution, collagen solution and natural extracellular matrix ECM solution.
The micro-channel solution in the step 4) is a silk fibroin solution, and the silk fibroin solution is prepared by adopting the following method:
4.1) degumming treatment method of natural silk: putting the purchased natural mulberry silk raw material into 0.02mol/LNa2CO3Decocting in the solution for 3 times, 30 min/time; fully washing with tap water to remove sericin, washing with deionized water twice to remove residual impurities, completely drying in a constant-temperature forced air drying oven at 50 ℃, and storing for later use;
4.2) preparing silk fibroin solution: accurately weighing 20g of degummed silk by an electronic balance; ② the degummed silk is cut into pieces and then dissolved in 9.3mol/L LiBr solution; thirdly, fully dissolving the mixture for 3 to 5 hours on a heat collection type magnetic stirrer at the temperature of 60 ℃; fourthly, continuously dialyzing the mixture for 2 days by using tap water, and intercepting the molecular weight to be 12000; dialyzing with deionized water for 1 day; sixthly, centrifuging for 10min at 4 ℃ at 10000rpm to remove impurities; seventhly, concentrating the 20 percent PEG solution for 12 hours, and keeping the molecular weight at 3500; eighty percent (10000 rpm) centrifugation is carried out at 4 ℃ for 10min to further remove impurities; ninthly, determining the concentration of the silk fibroin solution by a drying constant weight method and diluting the silk fibroin solution to the mass concentration of 10-20%; the r is stored in a refrigerator at 4 ℃ for later use.
5.3) fully soaking, eluting and leaching the fiber scaffold material in the composite scaffold by using an organic solvent, and specifically comprising the following steps of: soaking in dichloromethane for 2 days twice per day; then soaked in chloroform for 1 day, finally in chloroform: and (3) soaking the mixed solution in which the absolute methanol is mixed according to the volume ratio of 1:1 for 1 day, and sufficiently eluting and leaching the fiber scaffold material in the silk scaffold.
The material for preparing the long tubular fiber ring pore-forming scaffold template in the step 1) is Polycaprolactone (PCL), polyglycolic acid (PGA), polylactic acid-polyglycolic acid copolymer (PLGA) or Polyurethane (PU).
The invention also comprises the bionic microchannel integrated intervertebral disc bracket obtained by the preparation method and application thereof.
Compared with the prior art, the invention has the beneficial effects that:
1. the intervertebral disc scaffold obtained by preparing the micro-channel template and the nucleus pulposus template and then filling the micro-channel solution has two tissue structures of a bionic reconstruction fibrous ring and a nucleus pulposus, so that an integrated bionic micro-channel intervertebral disc scaffold is formed; provides a new method for realizing biological repair of severely degenerated intervertebral disc tissues.
2. The invention provides a supporting space for the growth of cells and tissues through the micro-channel structure, realizes the topological guide effect on the cells and the tissues, and provides a delivery carrier for the load of the cells or bioactive factors. Since the annulus fibrosus and nucleus pulposus have different microchannels, they have different topologically guiding effects on the growth of the tissue cells.
Drawings
FIG. 1 is a flow chart of preparation of a bionic microchannel silk fibroin integrated scaffold;
FIG. 2 is a diagram of a bionic microchannel silk fibroin integrated intervertebral disc scaffold, wherein, in the visual observation A, B, C are respectively a cross section and a sagittal plane image of a body microscope;
FIG. 3 is a SEM image of a biomimetic micro-channel intervertebral disc scaffold; the upper part is a transverse section picture, and the lower part is a sagittal section picture;
FIG. 4 is a 3-day Live/dead staining observation image of the bionic microchannel intervertebral disc scaffold inoculated cells; a, B, C area of annulus fibrosus (AF area); D. e, F is the nucleus pulposus region (NP Area);
FIG. 5 is a diagram showing the results of the cell proliferation assay of CCK-8;
FIG. 6 is a graph of cell spreading topography observed by phalloidin staining.
Detailed Description
In order to make the technical solutions of the present invention better understood by those skilled in the art, the present invention will be further described in detail with reference to the accompanying drawings and preferred embodiments.
Example 1: 1. preparation of experimental reagent
(1)0.02M Na2CO3Solution: weighing 2.12g of Na by an electronic balance2CO3And adding 800mL of deionized water into a 1L beaker to fully dissolve the solid, cooling to room temperature, transferring the liquid into a 1L volumetric flask, and slowly supplementing the deionized water to the 1L scale mark.
(2)9.3M LiBr (lithium bromide) solution: 60mL of deionized water is added into a 200mL beaker in advance, 80g of LiBr crystal is weighed by an electronic balance, then the LiBr crystal is slowly added into the beaker, a glass rod is used for stirring at the same time, the LiBr crystal is cooled to room temperature after being fully dissolved, and the deionized water is supplemented to 100mL of scale marks.
(3) 20% PEG (polyethylene glycol) solution: 600g of PEG solid is weighed by an electronic balance, transferred into a 3L measuring cylinder, added with 2L of deionized water, uniformly stirred by a glass rod, dissolved by a magnetic stirrer, and then fixed to a 3L scale mark by deionized water after the dissolution is completed, and stored in a refrigerator at 4 ℃ for later use.
(4) 0.5% PVA (polyvinyl alcohol) solution: 10g of PVA solid is weighed and dissolved in 2000mL of deionized water, and stirred at 60 ℃ until the PVA solid is completely dissolved for later use.
2. Preparing a bionic microchannel pore-forming template of a fibrous ring tissue:
(1) placing a PCL raw material in a high-temperature melting reaction furnace of a melt spinning machine, preheating at 180 ℃, and completely melting the PCL into a liquid state under the high-temperature condition;
(2) the parameters of a control panel of the melt spinning machine are set as follows: the temperature of the melting reaction furnace is 200 ℃, the rotating speed of a receiving rod is 120rpm, and the moving speed of a horizontal workbench is 60 Hz;
(3) adjusting the propelling speed of the micro-injection pump to be 2 mL/min;
(4) the diameter of the receiving rod is selected to be 2-14mm, the receiving distance is adjusted to be 1.5cm, and the specification of the syringe needle is 16G;
(5) the molten PCL liquid is slowly extruded out by a metal syringe needle, the PCL in the liquid state is uniformly drawn and then successfully wound on a rotating shaft center with the diameter of 2-14mm, and after the shaft center is removed, a long tubular fiber ring pore-forming template support with proper inner diameter and outer diameter and 60-degree oriented crossed and laminated arrangement of PCL fibers is obtained.
3. Preparing a nucleus pulposus tissue bionic porous structure pore-forming material (paraffin microspheres):
the paraffin wax microsphere is prepared by adopting a dispersion solidification method, which comprises the following specific steps:
(1) accurately weighing 60g of paraffin block, adding the paraffin block into 1200mL of 0.5% PVA solution, placing the solution on a constant-temperature magnetic stirrer, continuously stirring the solution at the temperature of 60 ℃, and uniformly dispersing the paraffin into paraffin microspheres after the paraffin is completely melted;
(2) after heating is stopped, continuously maintaining the stirring state, quickly adding ice water, and continuously stirring for a plurality of minutes to solidify and form the emulsified paraffin wax microspheres;
(3) size sorting is carried out by sieves with the aperture gradient of 40-60 meshes, 60-80 meshes, 80-100 meshes, 100-120 meshes and 120-140 meshes respectively, PVA is removed by fully washing with running water, and the dried product is dried after the alcohol dehydration;
(4) the paraffin wax microspheres with different grain diameters are prepared by the method, and are stored in a refrigerator at 4 ℃ for later use after being subpackaged.
4. Assembling a complete intervertebral disc bionic microchannel pore-forming template:
(1) placing a long tubular PCL fiber ring pore-forming template support prepared by a melt spinning technology in a constant-temperature air-blast drying oven to be heated for 10min at 60 ℃; (2) cutting the product into 1cm length with a blade after cooling; (3) sleeving a silicone tube with the inner diameter of 4mm on the outer layer of the fiber ring pore-forming template bracket; (4) filling paraffin microspheres (with the size of 80-120 meshes) in the centers of the tubular PCL pore-forming template supports; (5) heating at 55 deg.C for 20 min; (6) cooling to room temperature, and completing the preparation of the complete intervertebral disc pore-forming template.
5. Extracting a silk fibroin solution:
(1) the degumming treatment method of the natural silk comprises the following steps: putting the purchased natural mulberry silk raw material into 0.02mol/LNa2CO3Boiling the solution for 3 times (30 min/time), fully washing with tap water to remove sericin, washing with deionized water for two times to remove residual impurities, completely drying in a constant temperature forced air drying oven at 50 deg.C, and storing for use.
(2) Preparing a silk fibroin solution: firstly, accurately weighing 20g of degummed silk → secondly, cutting the degummed silk into pieces and dissolving the pieces in 9.3mol/L LiBr solution by using an electronic balance; thirdly, fully dissolving the mixture for 3 to 5 hours on a heat collection type magnetic stirrer at the temperature of 60 ℃; dialysis is carried out on tap water for 2 days (cut-off molecular weight is 12000); dialyzing with deionized water for 1 day; sixthly, centrifuging for 10min at 4 ℃ at 10000rpm to remove impurities; seventhly, concentrating the 20 percent PEG solution for 12 hours (with the molecular weight cutoff of 3500); eighty percent (10000 rpm) centrifugation is carried out at 4 ℃ for 10min to further remove impurities; ninthly, determining the concentration of the silk fibroin solution by a drying constant weight method; the r is stored in a refrigerator at 4 ℃ for later use.
6. Injecting a silk fibroin solution into the bionic microchannel pore-forming template:
(1) placing the prepared complete intervertebral disc bionic microchannel pore-forming template in a 1.5mL EP tube;
(2) adding a silk fibroin solution with the mass concentration of 10% -20% extracted in advance into an EP (EP) tube, and completely soaking a pore-forming template in the silk fibroin solution;
(3) repeatedly sucking the silk fibroin solution by using a vacuum drying pump under negative pressure to promote the silk fibroin solution to be fully poured into the internal gap of the complete pore-forming template;
(4) after the internal clearance of the pore-forming template is completely infiltrated by the silk fibroin solution, quickly transferring the EP tube into liquid nitrogen for freezing and forming;
(5) the freeze-formed silk fibroin scaffolds were transferred to a vacuum cryodryer for freeze-drying at low temperature (-60 ℃) for 3 days.
7. Elution leaching of template material in fibroin scaffolds:
(1) removing redundant silk fibroin materials at the periphery of the silk fibroin scaffold subjected to freeze drying and an outer layer silicone tube to obtain an SF/PCL/paraffin composite scaffold;
(2) soaking the SF/PCL/paraffin composite bracket in absolute ethyl alcohol for 2h to denature the silk fibroin;
(3) dichloromethane soak for 2 days (twice/day), then chloroform soak for 1 day, and finally chloroform: soaking the mixed solution of anhydrous methanol (1:1) for 1 day, and sufficiently eluting and leaching the PCL material in the fibroin bracket;
(4) fully washing the bracket by absolute ethyl alcohol to remove residual reagent;
(5) leaching the paraffin material by eluting with n-hexane by means of a soxhlet extractor;
(6) removing residual n-hexane by vacuum drying pump negative pressure suction;
(7) cutting the bracket which is sufficiently eluted and leached out of the template material into a thickness of 1mm by using a blade, and finishing the preparation of the bionic microchannel intervertebral disc integrated bracket. (as shown in fig. 2) and stored for later use.
Bionic microchannel integrated intervertebral disc scaffold factor load
(1) Performing radiation sterilization treatment on the stent by using radioactive isotope cobalt 60(Co60) with the dose of 15 Gy;
(2) dissolving the cytokine CCL5 in sterile deionized water according to the required concentration;
(3) then, pretreating the cut bionic microchannel integrated intervertebral disc bracket by using a heparin solution;
(4) soaking the heparinized bionic microchannel integrated intervertebral disc scaffold in a cell factor CCL5 solution, and putting the disc scaffold in a refrigerator at 4 ℃ overnight to facilitate the scaffold to fully adsorb factors;
(5) the scaffold loaded with the cytokines is stored in a refrigerator at the temperature of-20 ℃ for standby after being frozen and dried.
The application (loaded cells) of the bionic microchannel integrated intervertebral disc scaffold comprises the following specific operation steps:
the method comprises the following steps: pretreating the bionic microchannel integrated intervertebral disc scaffold:
in order to facilitate observation of migration and growth conditions of cells in the stent, the bionic microchannel silk fibroin integrated intervertebral disc stent is cut into a cylinder with the thickness of 2 mm. Radiation sterilization treatment with 15Gy radioactive isotope cobalt 60(Co60), pre-cell-seeded scaffoldSoaking in sterile PBS solution for 12 hr for fully hydrating, soaking in Fetal Bovine Serum (FBS) for 12 hr for fully adsorbing nutritional factors in serum, transferring the scaffold to 48-well cell culture plate containing DMEM before planting cells, and placing at 37 deg.C with 5% CO2The cells were cultured in the constant-temperature cell culture chamber of (1) for two hours in advance.
Step two: inoculating the bionic microchannel integrated intervertebral disc scaffold cells:
digesting 3T3 cells with good growth state into single cells by pancreatin/EDTA, counting, adjusting the cell suspension density to 1 × 106/mL, absorbing the liquid in the bracket by gauze in advance, uniformly inoculating 20 μ L of 3T3 cell suspension prepared in advance on the surface of the bracket by using a micropipette in a 48-hole cell culture plate, incubating for 3 hours in a constant-temperature cell culture box to accelerate the adhesion of the cells on the surface of the bracket, then supplementing 1mL of DMEM containing 10% FBS into each culture hole of the 48-hole cell culture plate for continuous culture, observing the color of the culture solution every day and changing the culture solution periodically.
Step three: bionic microchannel integrated intervertebral disc scaffold cell activity and proliferation detection
(1) Live/dead staining for cell viability: after the cell-inoculated scaffold is cultured for 3d, the cell-inoculated scaffold is taken out, washed for 3 times by sterile PBS (sterile PBS), soaked for 5min each time, added with prepared Live/dead staining working solution (20 muL and 5 muL of each of EthD-1 staining working solution and Calcein AM staining working solution are respectively dissolved in 10mL Hanks solution, and the solution is placed at 37 ℃ and contains 5 percent CO2The constant-temperature cell culture box is dyed for 30 minutes in a dark place, then a confocal microscope is used for observing the survival condition of cells in the bracket, the living cells are bright white and are adhered, spread and grown on the surface of the inner wall of the micro-channel; dead cells are in the shape of bright gray spheres and are attached to the inner wall of the microchannel. (as shown in FIG. 4)
(2) CCK-8 method for determining cell proliferation potency: digesting 3T3 cells with good growth state into single cells, and adjusting the concentration of cell suspension to 1 × 105Per mL; sucking internal liquid of a bionic microchannel intervertebral disc bracket and a control group bracket which are treated by FBS in advance, and placing the bionic microchannel intervertebral disc bracket and the control group bracket in a 96-hole cell culture plate; using micropipettor for aspiration20 μ L of 3T3 cell suspension was uniformly inoculated onto the surfaces of both sets of scaffolds, and placed at 37 ℃ with 5% CO2The incubation time in the constant-temperature cell incubator is 3 hours to accelerate the adhesion of cells on the surface of the bracket; supplementing 200mL of DMEM containing 10% FBS into each culture well of the 96-well cell culture plate for continuous culture, and regularly observing the color of the culture solution and changing the solution; completely absorbing the culture solution of the cell-inoculated scaffold after culturing for 1, 4 and 7 days respectively, then adding 100 mu L of culture solution containing 10% CCK-8 working solution into each hole, and putting the cells in a constant-temperature incubator for incubation for 2 hours in a dark place; each well draws 100. mu.L of the liquid, transfers it to a blank 96-well cell culture plate (this process is performed without generating air bubbles), and detects the absorbance (OD value) at a wavelength of 450nm of each well liquid using a microplate reader. CCK-8 cell proliferation detection results show that the OD values of the bionic microchannel bracket group and the control bracket group are in obvious increasing trend along with the prolonging of the culture time of the bracket-cell compound, which indicates that cells can survive on the two brackets and keep good proliferation capacity; the OD value of the bionic microchannel intervertebral disc bracket at each time point is higher than that of a control group, although the difference of the OD values of the bracket-cell complex cultured for 1 day is not statistically significant (P is more than 0.05), the difference of the OD values of the bracket-cell complex cultured for 4 days and 7 days is obvious, and the statistical significance is achieved (P is less than 0.05). The result shows that the bionic microchannel intervertebral disc bracket provides better space support for cell growth, and is beneficial to cell proliferation and internal migration. (as shown in FIG. 5)
(3) And (3) observing the cell spreading morphology by using phalloidin staining: taking out a sample to be tested, and soaking the sample for 3 times by using PBS (phosphate buffer solution), wherein each time is 5 minutes; fixing with 4% paraformaldehyde tissue fixing liquid for 15 minutes; washing with PBS for 3 times, and soaking for 5min each time; adding phalloidin staining solution (Phal loidin: PBS 1:1000), soaking the sample in the staining solution, and placing at 37 deg.C with 5% CO2The constant temperature cell culture box is dyed for 1 hour in a dark place; washing with PBS for 3 times, and soaking for 5min each time; the sample is placed in DAPI staining solution for dip-staining for 30 minutes to carry out color development observation on cell nucleuses; washing with PBS for 3 times, and soaking for 5min each time; and (3) observing the sample carried by the confocal cuvette under a confocal fluorescence microscope, and collecting and analyzing images. FITC-phalloidin staining results show that the bionic microchannel intervertebral disc scaffold can be used for treating the peripheral annulus fibrosus regionSee that a large number of cells adhered to the inner wall surface of the circular microchannel (see fig. 6A, B, C), the high magnification image shows that the cytoskeleton extends in a long fusiform along the inner wall of the channel (see fig. 6D); the central nucleus pulposus region shows that a large number of cells are adhered to and grow on the inner surface of the spherical pore canal, a large number of pseudo feet are fused and grown among the cells (see fig. 6E, F and G), the high magnification image shows that cytoskeleton is adhered to and randomly grows along the inner wall of the spherical pore canal, and the morphology of the cytoskeleton has no obvious orientation (see fig. 6H). (as shown in fig. 6), it can be seen that different microstructures of the biomimetic scaffold can guide cells to present different growth forms, and have good topological guiding effect on tissue regeneration and remodeling.
The invention discloses a bionic microchannel integrated intervertebral disc biological scaffold, which is used for bionic reconstruction of a collagen fiber oblique crossing laminated arrangement structure in an annulus fibrosus tissue and bionic reconstruction of a nucleus pulposus tissue porous spongy extracellular matrix structure. The micro-channels in the fiber ring area have the characteristic of directional cross arrangement, the micro-channels have good connectivity, and cells can be well adhered to the surface of the inner wall of the micro-channels and show the framework form of directional growth; the micro-channel in the nucleus pulposus region has a porous structure with a high communication characteristic, and cells are easy to migrate and grow to the inside of the bracket after being adhered to the inner wall of the pore channel. The support structure highly simulates the microstructure of a natural intervertebral disc and provides good support guiding effect for the artificial natural regeneration of the intervertebral disc. The applicant designs a pure bracket without a micro-channel, namely a solid integrated intervertebral disc biological bracket; the scaffold cell tissue without the micro-channel structure is difficult to grow into the scaffold, is not beneficial to tissue regeneration, and has no topological guide effect on tissue remodeling.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A preparation method of a bionic microchannel integrated intervertebral disc stent is characterized by comprising the following steps:
1) preparing a bionic microchannel pore-forming template of a fibrous ring tissue: preparing fibers with the inner diameter of 2-8mm and the outer diameter of 4-14mm by using a melt spinning technology; the fiber is in 60 degrees, oriented, crossed and laminated arrangement with the long tubular fiber ring pore-forming scaffold template;
2) preparing a pore-forming material with a bionic porous structure of nucleus pulposus tissue: preparing nucleus pulposus template microspheres by adopting a dispersion solidification method;
3) assembling a complete intervertebral disc bionic microchannel pore-forming template: 3.1) drying and cutting the long tubular fiber ring pore-forming support template obtained in the step 1); 3.2) sleeving a silicone tube on the outer layer of the cut fiber ring pore-forming scaffold template; 3.3) filling the center of the fiber ring pore-forming template bracket sleeved with the silicone tube with the nucleus pulposus template microspheres obtained in the step 2);
4) pouring a microchannel solution into the bionic microchannel pore-forming template: completely soaking the complete bionic microchannel pore-forming template of the intervertebral disc obtained in the step 3) in a microchannel solution, and repeatedly sucking the microchannel solution by using a vacuum drying pump under negative pressure to promote the microchannel solution to be fully filled into the internal gap of the complete pore-forming template; freezing, molding and drying;
5) elution leaching of template material in fibroin scaffolds: 5.1) removing redundant silk fibroin materials at the periphery of the freeze-dried silk fibroin bracket and an outer layer silicone tube to obtain a silk fibroin/fiber bracket/nucleus pulposus template composite bracket; 5.2) soaking the silk fibroin/fiber scaffold/nucleus pulposus template composite scaffold in absolute ethyl alcohol to denature the silk fibroin; 5.3) fully soaking, eluting and leaching the fiber scaffold material in the composite scaffold by using an organic solvent; 5.4) fully washing the bracket by absolute ethyl alcohol to remove residual reagent; 5.5) leaching the nucleus pulposus template material by using n-hexane for elution by a soxhlet extractor; 5.6) removing residual n-hexane by vacuum drying pump negative pressure suction; storing for later use.
2. The preparation method of the bionic microchannel integrated intervertebral disc scaffold as claimed in claim 1, wherein the step 1) comprises the following specific steps:
1.1) putting the fiber support material into a high-temperature melting reaction furnace of a melt spinning machine, and completely melting the fiber support material into a liquid state;
1.2) setting parameters of a control panel of the melt spinning machine as follows: the temperature of the melting reaction furnace is 180-210 ℃, the rotating speed of the receiving rod is 50-150rpm, and the moving speed of the horizontal workbench is 50-100 Hz; adjusting the propelling speed of the micro-injection pump to be 1-5 mL/min; selecting the diameter of the receiving rod to be 2-14mm, adjusting the receiving distance to be 1-3cm, and adjusting the specification of the syringe needle to be 14-20G;
1.3) slowly extruding the molten fiber support material liquid by a metal syringe needle, uniformly drawing the fiber support material in a liquid state, successfully winding the fiber support material on a rotating shaft center with the diameter of 2-14mm, and removing the shaft center to obtain a long tubular fiber ring pore-forming support template with proper inner diameter and outer diameter and fibers in 60-degree oriented crossed laminated arrangement.
3. The preparation method of the bionic microchannel integrated intervertebral disc scaffold as claimed in claim 2, wherein the parameters of the control panel of the melt spinning machine set in the step 1.2) are as follows: the temperature of the melting reaction furnace is 200 ℃, the rotating speed of a receiving rod is 120rpm, and the moving speed of a horizontal workbench is 60 Hz; adjusting the propelling speed of the micro-injection pump to be 2 mL/min; the diameter of the receiving rod is selected to be 2-14mm, the receiving distance is adjusted to be 1.5cm, and the specification of the syringe needle is 16G.
4. The preparation method of the bionic microchannel integrated intervertebral disc scaffold as claimed in claim 1, wherein the nucleus pulposus template microspheres are paraffin microspheres, and the specific method is as follows:
2.1) accurately weighing 60 parts by mass of paraffin blocks, adding the paraffin blocks into a PVA solution containing 1200 parts by volume of 0.5 wt%, placing the mixture on a constant-temperature magnetic stirrer, and continuously stirring the mixture at the temperature of 60 ℃ until the paraffin is completely melted and uniformly dispersed in the PVA solution to form paraffin microspheres;
2.2) continuing to maintain the stirring state after stopping heating, quickly adding ice water, and continuing to stir for a plurality of minutes to solidify and form the emulsified paraffin wax microspheres;
2.3) carrying out size sorting by using sieves with the aperture gradients of 40-60 meshes, 60-80 meshes, 80-100 meshes, 100-140 meshes and 120-140 meshes, fully washing with running water to remove PVA, and drying after alcohol dehydration;
2.4) preparing the paraffin microspheres with different grain diameters by the method, subpackaging and storing in a refrigerator at 4 ℃ for later use.
5. The preparation method of the bionic microchannel integrated intervertebral disc scaffold as claimed in claim 1, wherein the specific steps of the step 3) are as follows: 3.1) placing the long tubular fiber ring pore-forming support template prepared by the melt spinning technology in a constant-temperature air-blast drying oven to be heated for 10min at 60 ℃; cutting the product into 1cm length with a blade after cooling; 3.2) sleeving a silicone tube with a proper inner diameter on the outer layer of the long tubular fiber ring pore-forming scaffold template; 3.3) nucleus pulposus template microspheres with the size of 80-120 meshes are filled in the center of the long tubular fiber ring pore-forming stent template; heating at 55 deg.C for 20 min; cooling to room temperature, and completing the preparation of the complete intervertebral disc pore-forming template.
6. The method for preparing the bionic microchannel integrated intervertebral disc scaffold as claimed in claim 1, wherein the microchannel solution in step 4) is silk fibroin solution, chitosan solution, collagen solution, natural extracellular matrix ECM solution.
7. The preparation method of the bionic microchannel integrated intervertebral disc scaffold as claimed in claim 6, wherein the microchannel solution in the step 4) is a silk fibroin solution;
the silk fibroin solution is prepared by adopting the following method:
4.1) degumming treatment method of natural silk: putting the purchased natural mulberry silk raw material into 0.02mol/LNa2CO3Decocting in the solution for 3 times, 30 min/time; fully washing with tap water to remove sericin, washing with deionized water twice to remove residual impurities, completely drying in a constant-temperature forced air drying oven at 50 ℃, and storing for later use;
4.2) preparing silk fibroin solution: accurately weighing 20g of degummed silk by an electronic balance; ② the degummed silk is cut into pieces and then dissolved in 9.3mol/L LiBr solution; thirdly, fully dissolving the mixture for 3 to 5 hours on a heat collection type magnetic stirrer at the temperature of 60 ℃; fourthly, continuously dialyzing the mixture for 2 days by using tap water, and intercepting the molecular weight to be 12000; dialyzing with deionized water for 1 day; sixthly, centrifuging for 10min at 4 ℃ at 10000rpm to remove impurities; seventhly, concentrating the 20 percent PEG solution for 12 hours, and keeping the molecular weight at 3500; eighty percent (10000 rpm) centrifugation is carried out at 4 ℃ for 10min to further remove impurities; ninthly, determining the concentration of the silk fibroin solution by a drying constant weight method and diluting the silk fibroin solution to the mass concentration of 10-20%; the r is stored in a refrigerator at 4 ℃ for later use.
8. The preparation method of the bionic microchannel integrated intervertebral disc scaffold as claimed in claim 1, wherein 5.3) the step of sufficiently soaking, eluting and leaching the fiber scaffold material in the composite scaffold by using an organic solvent specifically comprises the following steps: soaking in dichloromethane for 2 days twice per day; then soaked in chloroform for 1 day, finally in chloroform: and (3) soaking the mixed solution in which the absolute methanol is mixed according to the volume ratio of 1:1 for 1 day, and sufficiently eluting and leaching the fiber scaffold material in the silk scaffold.
9. A bionic microchannel integrated intervertebral disc scaffold obtained by the preparation method of any one of claims 1 to 8.
10. Use of the biomimetic microchannel integrated disc scaffold of claim 9.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114681688A (en) * | 2022-04-15 | 2022-07-01 | 东南大学 | Tissue regeneration membrane for promoting vascularization by using micro-channel and preparation method thereof |
| CN115836932A (en) * | 2023-02-16 | 2023-03-24 | 首都医科大学附属北京天坛医院 | Personalized bionic artificial cervical intervertebral disc prosthesis manufacturing method and prosthesis |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1718173A (en) * | 2005-03-14 | 2006-01-11 | 西安交通大学 | Double-layered bionic pseudo-structure of cervical intervertebral disci, and its mfg. process |
| KR100744833B1 (en) * | 2006-02-24 | 2007-08-01 | 전북대학교산학협력단 | Artificial intervertebral disc comprising disc cell and the method of tissue engineered disc regeneration using the said disc cell |
| KR20150044686A (en) * | 2013-10-17 | 2015-04-27 | 단국대학교 산학협력단 | Polymer Nanofiber scaffold for osteochondral regeneration |
| CN108498870A (en) * | 2018-06-08 | 2018-09-07 | 南京邦鼎生物科技有限公司 | A kind of complete Biotype artificial interverbebral disc and preparation method thereof |
| CN109999227A (en) * | 2019-03-28 | 2019-07-12 | 武汉大学 | A kind of preparation method and application based on fibroin albumen and the embedded hydrogel cartilage biomimetic scaffolds of the blended nanofiber of chitin |
| CN110507853A (en) * | 2019-09-05 | 2019-11-29 | 上海交通大学医学院附属第九人民医院 | A kind of method of biological 3 D-printing interverbebral disc in support liquid medium |
| CN113040982A (en) * | 2021-03-03 | 2021-06-29 | 中山大学 | Bionic fiber ring stent and preparation method thereof |
-
2021
- 2021-07-14 CN CN202110792993.0A patent/CN113576719A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1718173A (en) * | 2005-03-14 | 2006-01-11 | 西安交通大学 | Double-layered bionic pseudo-structure of cervical intervertebral disci, and its mfg. process |
| KR100744833B1 (en) * | 2006-02-24 | 2007-08-01 | 전북대학교산학협력단 | Artificial intervertebral disc comprising disc cell and the method of tissue engineered disc regeneration using the said disc cell |
| KR20150044686A (en) * | 2013-10-17 | 2015-04-27 | 단국대학교 산학협력단 | Polymer Nanofiber scaffold for osteochondral regeneration |
| CN108498870A (en) * | 2018-06-08 | 2018-09-07 | 南京邦鼎生物科技有限公司 | A kind of complete Biotype artificial interverbebral disc and preparation method thereof |
| CN109999227A (en) * | 2019-03-28 | 2019-07-12 | 武汉大学 | A kind of preparation method and application based on fibroin albumen and the embedded hydrogel cartilage biomimetic scaffolds of the blended nanofiber of chitin |
| CN110507853A (en) * | 2019-09-05 | 2019-11-29 | 上海交通大学医学院附属第九人民医院 | A kind of method of biological 3 D-printing interverbebral disc in support liquid medium |
| CN113040982A (en) * | 2021-03-03 | 2021-06-29 | 中山大学 | Bionic fiber ring stent and preparation method thereof |
Non-Patent Citations (5)
| Title |
|---|
| 丁晓明: "含软骨钙化层的仿生骨软骨支架复合脂肪干细胞构建组织工程骨软骨的实验研究", 《硕士电子期刊出版信息》, pages 6 * |
| 张同星: "仿生微通道丝素蛋白一体化支架用于椎间盘退变再生修复的研究", 《中国博士学位论文全文数据库医药卫生科技辑》, pages 6 - 8 * |
| 曾超;朱美峰;徐宝山;杨强;王连永;李秀兰;张杨;夏群;许海委;伍耀宏;: "丝素蛋白多孔支架与兔髓核细胞生物相容性研究", 中国矫形外科杂志, no. 23 * |
| 李麒峰;徐宝山;杨强;马信龙;张杨;郭悦;杨阳;张凯辉;夏金健;张维昊;: "聚己内酯/海藻酸钠/壳聚糖材料制备组织工程椎间盘双相支架", 天津医药, no. 04 * |
| 杜立龙: "丝素蛋白来源的一体化纤维环-髓核支架的制备与评估", 天津医药, vol. 43, no. 6, pages 603 - 606 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114681688A (en) * | 2022-04-15 | 2022-07-01 | 东南大学 | Tissue regeneration membrane for promoting vascularization by using micro-channel and preparation method thereof |
| CN115836932A (en) * | 2023-02-16 | 2023-03-24 | 首都医科大学附属北京天坛医院 | Personalized bionic artificial cervical intervertebral disc prosthesis manufacturing method and prosthesis |
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