CN113750291A - Articular cartilage scaffold and preparation method thereof - Google Patents
Articular cartilage scaffold and preparation method thereof Download PDFInfo
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- CN113750291A CN113750291A CN202111210839.4A CN202111210839A CN113750291A CN 113750291 A CN113750291 A CN 113750291A CN 202111210839 A CN202111210839 A CN 202111210839A CN 113750291 A CN113750291 A CN 113750291A
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Abstract
The invention provides an articular cartilage support and a preparation method thereof, wherein the preparation method comprises the following steps: obtaining a two-dimensional nanofiber membrane through electrostatic spinning; preparing a hyaluronic acid solution; dipping the two-dimensional nanofiber membrane in the hyaluronic acid solution to obtain a two-dimensional nanofiber membrane of cross-linked hyaluronic acid; foaming the two-dimensional nanofiber membrane of the cross-linked hyaluronic acid to obtain a three-dimensional nanofiber scaffold; and drying the three-dimensional nanofiber scaffold to obtain the articular cartilage scaffold with a multilayer three-dimensional structure. According to the preparation method provided by the invention, the articular cartilage scaffold with a multilayer three-dimensional structure is prepared by combining electrostatic spinning, chemical crosslinking and gas foaming; the cartilage support has a porous structure inside, is good in bulkiness, is crosslinked with hyaluronic acid, has the biomechanical performance and the biological functionality matched with articular cartilage, and is beneficial to increasing the control on the defect shape of the cartilage so as to better fill the defect.
Description
Technical Field
The invention relates to the technical field of biomedical materials, in particular to an articular cartilage support and a preparation method thereof.
Background
Articular cartilage is connective tissue that covers the epiphyseal surface of the articular bone, without blood or lymphatic vessels, and therefore has a limited ability to self-heal. Articular cartilage is composed of chondrocytes, a mature, highly specialized cell type responsible for the renewal of the extracellular matrix (ECM), but very slowly, and extracellular matrix. The clinical incidence of articular cartilage defects is extremely high, which is one of the main causes of limb disability and causes huge economic loss to the society.
Trauma or degenerative disease often results in the gradual degeneration of tissues, resulting in joint pain, functional impairment, and degenerative arthritis. For the generally minor cartilage lesions, microfracture techniques can be used for treatment, but microfracture techniques often result in the formation of fibrocartilage, which is biochemically and biomechanically inferior to native hyaline articular cartilage. The limitations of microfracture techniques have also promoted the development of autologous chondrocyte transplantation, but autologous cartilage transplantation techniques require two surgeries and longer recovery times, and thus these techniques are not long-term clinical solutions.
In recent years, with the development of tissue engineering technology, the realization of the repair and regeneration of articular cartilage defects by applying tissue engineering technology has received extensive attention and research. The electrostatic spinning is a reliable method for preparing the nanofiber tissue engineering scaffold with high porosity, suitable biomechanical property and biodegradation rate. However, the nanofiber membrane prepared by the traditional electrostatic spinning technology is compact, only has a porous structure on the surface, is not beneficial to the permeation of cells into the interior of the scaffold, and limits the application of the nanofiber membrane in the aspect of articular cartilage scaffolds.
Disclosure of Invention
The invention solves the problem that the nanofiber membrane prepared by the existing electrostatic spinning technology is compact and is not beneficial to the permeation of cells into the inside of the scaffold when being used for the articular cartilage scaffold.
In order to solve the above problems, the present invention provides a method for preparing an articular cartilage scaffold, comprising the steps of:
s1: dissolving degradable polymer and natural high molecular material in a solvent to prepare spinning solution;
s2: performing electrostatic spinning on the spinning solution through electrostatic spinning equipment to obtain a two-dimensional nanofiber membrane;
s3: preparing a hyaluronic acid solution;
s4: dipping the two-dimensional nanofiber membrane in the hyaluronic acid solution, and freeze-drying to obtain a two-dimensional nanofiber membrane of cross-linked hyaluronic acid;
s5: placing the two-dimensional nanofiber membrane of the cross-linked hyaluronic acid in a foaming agent, and foaming to obtain a three-dimensional nanofiber scaffold;
s6: and drying the three-dimensional nanofiber scaffold to obtain the articular cartilage scaffold with a multilayer three-dimensional structure.
Optionally, configuring the hyaluronic acid solution comprises:
s31: preparing MES buffer solution;
s32: adding hyaluronic acid into the MES buffer solution to obtain a MES buffer solution of the hyaluronic acid;
s33: and adding EDC/NHS into the MES buffer solution of the hyaluronic acid, and carrying out carboxyl activation reaction to obtain the hyaluronic acid solution.
Optionally, the blowing agent is an aqueous sodium borohydride solution.
Optionally, the foaming time is 5min to 30 min.
Optionally, in the spinning solution of step S1, the total mass concentration of the degradable polymer and the natural polymer material ranges from 8% to 15%.
Optionally, the mass ratio of the degradable polymer to the natural polymer material is 4: 1.
optionally, the degradable polymer is selected from one of polylactic acid-caprolactone copolymer and polycaprolactone.
Optionally, the natural polymer material is selected from one of silk fibroin and collagen.
Alternatively, the solvent in step S1 is selected from one of hexafluoroisopropanol and dichloromethane.
Another object of the present invention is to provide an articular cartilage scaffold prepared by the method for preparing an articular cartilage scaffold as described above.
Compared with the prior art, the preparation method of the articular cartilage scaffold provided by the invention has the following advantages:
according to the preparation method of the articular cartilage scaffold, the articular cartilage scaffold with a multilayer three-dimensional structure is prepared by combining electrostatic spinning, chemical crosslinking and gas foaming; the cartilage support with the multilayer three-dimensional structure has a porous structure inside, is good in bulkiness, and is crosslinked with hyaluronic acid, so that the biomechanical performance and the biological functionality of the cartilage support are matched with those of articular cartilage, the control on the defect shape of the cartilage is favorably increased, and the defect is filled better; and helps to reduce complications of autologous chondrocyte donor sites during use; and the preparation technology is simpler, and the stability of the implant is improved. In addition, in the using process of the cartilage scaffold, as the chondrocytes are cultured in a three-dimensional environment, the chondrocytes are not easy to differentiate, and hyaline cartilage is easier to generate, so that the articular cartilage scaffold has important significance in the repair and regeneration of articular cartilage defects.
Drawings
FIG. 1 is a photograph showing an articular cartilage scaffold in example 1 of the present invention, and b is a photograph showing an articular cartilage scaffold in example 2 of the present invention;
FIG. 2 is a scanning electron microscope showing the surface of the articular cartilage scaffold in example 1 of the present invention, and FIG. b is a scanning electron microscope showing the cross-section of the articular cartilage scaffold in example 1 of the present invention;
FIG. 3 is an infrared spectrum of hyaluronic acid, a three-dimensional scaffold of non-crosslinked hyaluronic acid and a three-dimensional scaffold of crosslinked hyaluronic acid in example 1 of the present invention;
FIG. 4 is a graph showing the compressive stress strain of the articular cartilage scaffold of example 1 of the present invention and the articular cartilage scaffold of comparative example 1.
Detailed Description
The following describes embodiments of the present invention in detail. The embodiments described below are exemplary and are intended to be illustrative of the present invention and should not be construed as limiting the present invention, and all other embodiments that can be obtained by one of ordinary skill in the art based on the embodiments of the present invention without inventive step fall within the scope of the present invention.
In order to solve the problem that the nanofiber membrane prepared by the existing electrostatic spinning technology is compact and is not beneficial to the permeation of cells into the inside of the scaffold when the nanofiber membrane is used for the articular cartilage scaffold, the invention provides a preparation method of the articular cartilage scaffold, which comprises the following steps:
s1: dissolving degradable polymer and natural high molecular material in a solvent to prepare spinning solution;
s2: performing electrostatic spinning on the spinning solution through electrostatic spinning equipment to obtain a two-dimensional nanofiber membrane;
s3: preparing a hyaluronic acid solution;
s4: dipping the two-dimensional nanofiber membrane in a hyaluronic acid solution, and drying to obtain a two-dimensional nanofiber membrane of cross-linked hyaluronic acid;
s5: placing the two-dimensional nanofiber membrane of the cross-linked hyaluronic acid in a foaming agent, and foaming to obtain a three-dimensional nanofiber scaffold;
s6: and drying the three-dimensional nanofiber scaffold to obtain the articular cartilage scaffold with a multilayer three-dimensional structure.
The method comprises the steps of firstly, preparing a two-dimensional nanofiber membrane with a two-dimensional structure by using a degradable polymer and a natural high polymer material as raw materials through an electrostatic spinning method; the preparation method of the two-dimensional nanofiber membrane can adopt a traditional electrostatic spinning method for preparation; the electrostatic spinning method is preferably a dynamic water flow electrostatic spinning method or a roller receiving electrostatic spinning method; and preferably, the spinning parameters in the electrostatic spinning process are as follows: the voltage is 10kV to 12kV, the receiving distance is 10cm to 15cm, and the flow rate of the spinning solution is 1.0ml/h to 1.2 ml/h.
Step S2, after electrostatic spinning, further comprises a drying process; the application preferably selects the drying process as vacuum drying, the vacuum degree in the drying process is-35 kPa to-25 kPa, the drying temperature is 25 ℃ to 30 ℃, and the drying time is 24h to 48 h.
Further preparing a hyaluronic acid solution, and placing the prepared two-dimensional nanofiber membrane in the hyaluronic acid solution for dipping so as to perform chemical crosslinking on the two-dimensional nanofiber membrane, thereby obtaining the two-dimensional nanofiber membrane of crosslinked hyaluronic acid; the nanofiber membrane has good elasticity and excellent biocompatibility through chemical crosslinking of hyaluronic acid, so that the nanofiber membrane of the crosslinked hyaluronic acid can better simulate the articular cartilage extracellular matrix in chemical composition.
The prepared two-dimensional nanofiber membrane is soaked in the hyaluronic acid solution, so that on one hand, hyaluronic acid can be crosslinked on the surface of nanofibers in the two-dimensional nanofiber membrane through a chemical crosslinking effect, cartilage cells can be conveniently contacted with the hyaluronic acid, biocompatibility of the articular cartilage scaffold can be improved, and the connection strength between the hyaluronic acid and the articular cartilage scaffold can be improved; on the other hand, in the dipping process, the hyaluronic acid crosslinked on the surface of the two-dimensional nanofiber can further generate a crosslinking reaction, so that the connection strength between the hyaluronic acid and the articular cartilage scaffold is further improved, and the stability of the articular cartilage scaffold structure and performance is improved.
In addition, the content of hyaluronic acid in the cross-linked articular cartilage scaffold can be regulated and controlled by adding the amount and time of hyaluronic acid in a reaction system, and a reaction medium is completely an aqueous solution, so that the cross-linked articular cartilage scaffold is non-toxic, high in processing efficiency and strong in repeatability; in the existing articular cartilage scaffold, hyaluronic acid is usually added into spinning solution, and is introduced in a direct blending mode, so that on one hand, the amount of the introduced hyaluronic acid is small, the introduced hyaluronic acid exists in a nanofiber mode, only one part of the introduced hyaluronic acid is positioned on the surface of the nanofiber, and the improvement degree of the biocompatibility of the articular cartilage scaffold is limited; on the other hand, the direct blending method takes a lot of time to stir to dissolve hyaluronic acid, and needs to further crosslink hyaluronic acid after the scaffold is obtained by electrostatic spinning, which is time-consuming and low in processing efficiency.
In order to enable the prepared articular cartilage scaffold to better simulate the articular cartilage extracellular matrix structurally, the two-dimensional nanofiber membrane of the cross-linked hyaluronic acid is placed in a foaming agent, and foaming is carried out, so that on one hand, the thickness of the two-dimensional nanofiber membrane of the cross-linked hyaluronic acid is increased, and the two-dimensional structure is converted into a multi-layer three-dimensional structure from the two-dimensional structure, and hydrogen is generated due to the reaction of sodium borohydride and water, and is continuously accumulated in the pores of the nanofibers and is accumulated along with the passage of time, so that the pores among the nanofibers are opened, and the multi-layer structure is formed; on the other hand, a porous structure is formed in the three-dimensional structure, and the nanofiber scaffold with the multilayer three-dimensional structure can be obtained after further freeze drying, wherein the nanofiber scaffold is the articular cartilage scaffold with the multilayer three-dimensional structure.
According to the preparation method of the articular cartilage scaffold, the articular cartilage scaffold with a multilayer three-dimensional structure is prepared by combining electrostatic spinning, chemical crosslinking and gas foaming; the cartilage support with the multilayer three-dimensional structure has a porous structure inside, is good in bulkiness, and is crosslinked with hyaluronic acid, so that the biomechanical performance and the biological functionality of the cartilage support are matched with those of articular cartilage, the control on the defect shape of the cartilage is favorably increased, and the defect is filled better; and helps to reduce complications of autologous chondrocyte donor sites during use; and the preparation technology is simpler, and the stability of the implant is improved. In addition, in the using process of the cartilage scaffold, as the chondrocytes are cultured in a three-dimensional environment, the chondrocytes are not easy to dedifferentiate and are easier to generate hyaline cartilage, so that the articular cartilage scaffold has important significance in the repair and regeneration of articular cartilage defects.
The preparation of the hyaluronic acid solution in step S3 of the present application includes the following steps:
s31: preparing 2- (N-morpholine) ethanesulfonic acid (MES) buffer solution;
s32: adding hyaluronic acid into the MES buffer solution to obtain the MES buffer solution of the hyaluronic acid;
s33: adding 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) into MES buffer solution of hyaluronic acid, and carrying out carboxyl group activation reaction to obtain hyaluronic acid solution.
In the reaction process, in order to ensure that the reaction is carried out smoothly, the 2- (N-morpholine) ethanesulfonic acid (MES) buffer solution is 2- (N-morpholine) ethanesulfonic acid aqueous solution, and preferably, in the process of preparing the MES buffer solution, the pH of the MES buffer solution is adjusted to about 5 by sodium hydroxide or potassium hydroxide; in the hyaluronic acid solution, hyaluronic acid is a cross-linking product, and EDC and NHS are chemical cross-linking agents; the time for the reaction of activating the carboxyl group in step S33 is preferably 20min to 50min, and more preferably 30 min.
The time for soaking the two-dimensional nanofiber membrane in the hyaluronic acid solution for chemical crosslinking is preferably 4-8 h, and the time for chemical crosslinking is further preferably 6 h; according to the preparation method, the degradable polymer and the natural high polymer material are used as raw materials to prepare the two-dimensional nanofiber membrane, and then the two-dimensional nanofiber membrane is placed in the hyaluronic acid solution to be chemically crosslinked, so that hyaluronic acid can be crosslinked on the degradable polymer and the natural high polymer in the two-dimensional nanofiber membrane, and the two-dimensional nanofiber membrane can form a multilayer three-dimensional structure after subsequent foaming while the nanofiber membrane has good elasticity and excellent biocompatibility due to chemical crosslinking of hyaluronic acid.
In the application, the drying process in the step S4 is preferably freeze drying, wherein in the freeze drying process, the vacuum degree is 0.5 Pa-1 Pa, the drying temperature is-80 ℃, and the drying time is 24 h-48 h.
Placing the two-dimensional nanofiber membrane of the cross-linked hyaluronic acid in a foaming agent for foaming, and impacting the two-dimensional nanofiber membrane by using gas generated by the foaming agent, so that the two-dimensional nanofiber membrane of the cross-linked hyaluronic acid is layered under the gas impact to form a multilayer three-dimensional structure; on the other hand, a porous structure appears in the nanofiber membrane, and the three-dimensional growth capacity of cells in the scaffold is improved.
The foaming agent is preferably sodium borohydride aqueous solution, so that hydrogen is released through the reaction of sodium borohydride and water, and the nanofiber membrane is impacted through the hydrogen, so that the nanofiber membrane is layered and forms a porous fluffy structure inside; specifically, as sodium borohydride reacts with water, hydrogen gas is continuously accumulated in pores of the nanofiber membrane, the amount of hydrogen gas is accumulated over time, and the accumulated hydrogen gas gradually opens the pores between the nanofibers, so that a multilayer structure is formed.
The thickness and the inner pore diameter of the prepared multilayer three-dimensional articular cartilage scaffold are related to the concentration of a foaming agent and the foaming time, the concentration of the foaming agent can be one of 0.01M, 0.1M and 1M, the concentration of the foaming agent is preferably 0.1M, the foaming time is 5-30 min, so that the thickness of the multilayer three-dimensional articular cartilage scaffold is controlled to be 0.5-5 mm, and the pore diameter of the inner porous structure is controlled to be 10-30 mu M.
Further freeze-drying the three-dimensional nanofiber scaffold obtained after foaming to obtain the articular cartilage scaffold with a multilayer three-dimensional structure; in the application, the drying process in the step S6 is preferably freeze drying, wherein in the freeze drying process, the vacuum degree is 0.5 Pa-1 Pa, the drying temperature is-80 ℃, and the drying time is 24 h-48 h.
The interlayer spacing of the multilayer three-dimensional articular cartilage scaffold is related to the thickness of the two-dimensional nanofiber membrane and the thickness of the multilayer three-dimensional articular cartilage scaffold, and the interlayer spacing of the multilayer three-dimensional articular cartilage scaffold is preferably 40-110 microns.
In order to enable the structure and performance of the multilayer three-dimensional articular cartilage scaffold to meet the use requirements, the total mass concentration range of the degradable polymer and the natural high molecular material in the spinning solution of the step S1 is preferably 8% -15%, and the total mass concentration of the degradable polymer and the natural high molecular material is further preferably 10%; the mass ratio of the degradable polymer to the natural high molecular material is 4: 1.
the degradable polymer and the natural polymer material in the step S1 can be selected according to the prior art, and in the present application, preferably, the degradable polymer is selected from one of polylactic acid-caprolactone copolymer and polycaprolactone, the natural polymer material is selected from one of silk fibroin and collagen, and the solvent in the step S1 is selected from one of hexafluoroisopropanol and dichloromethane.
The preparation method of the articular cartilage provided by the invention is simple to operate and good in repeatability, and provides a new idea for the design of the articular cartilage support.
Another object of the present invention is to provide an articular cartilage scaffold prepared by the method for preparing an articular cartilage scaffold as described above.
The articular cartilage scaffold provided by the invention has a multilayer three-dimensional structure, has a porous structure inside, and is crosslinked with hyaluronic acid, so that the articular cartilage scaffold has good chemical structure stability, good elasticity and excellent biocompatibility; the chemically crosslinked multilayer three-dimensional articular cartilage scaffold has improved mechanical properties without influencing biocompatibility, can promote cell infiltration, maintain chondrocyte phenotype for a long time and promote regeneration capacity of cartilage tissues, and is favorable for repairing articular cartilage injuries.
Compared with the existing articular cartilage support, the articular cartilage support provided by the invention has the advantages that the control of cartilage defect shapes is increased, so that defects can be filled better; the complication of the autologous chondrocyte supply area is reduced, and the stability of the graft is increased; in addition, since chondrocytes are cultured in a three-dimensional environment, chondrocytes are not easily dedifferentiated, and thus hyaline cartilage is more easily produced.
The thickness of the articular cartilage support is preferably 0.5-5 mm, the fiber diameter is 0.5-1.5 mu m, and the pore diameter is 10-30 mu m; the pore size range is larger than that of the traditional two-dimensional nanofiber membrane, so that cells can migrate into the interior of the scaffold; the interlayer spacing of the multilayer structure is 40-110 μm, so that the articular cartilage support can bear the stress of 3 kPa.
In conclusion, the articular cartilage scaffold provided by the invention has proper pore size and a multilayer structure, solves the problems of small pore size, dense fiber arrangement and difficult cell growth of the nanofiber scaffold, and improves the three-dimensional growth capacity of cells in the scaffold; after hyaluronic acid is chemically crosslinked, the elasticity and the structural stability of the scaffold are improved, so that the articular cartilage scaffold is closer to the physical and chemical compositions of the natural cartilage extracellular matrix, the phenotype of cartilage cells is maintained, and the expression of the cartilage extracellular matrix is promoted.
The multilayer nanofiber three-dimensional articular cartilage scaffold provided by the invention provides an articular cartilage tissue engineering scaffold with clinical application value, brings a new method and a new idea for treating clinical articular cartilage injury, and has the advantages of simple operation, good repeatability and high economic benefit.
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.
Example 1
This example provides a method for preparing an articular cartilage scaffold, which includes the following steps:
s1: preparing a spinning solution: weighing 0.8g of polylactic acid-polycaprolactone (P (LLA-CL)) with molecular weight of eight ten thousand and 0.2g of Silk Fibroin (SF) and dissolving in 10mL of Hexafluoroisopropanol (HFIP) to prepare P (LLA-CL)/SF spinning solution with mass concentration of 10%;
s2: preparing a two-dimensional nanofiber membrane: preparing a P (LLA-CL)/SF two-dimensional nanofiber membrane by dynamic water flow electrostatic spinning, and spinning by adopting a water circulation system and a device; the device consists of two basins, namely a top basin and a bottom basin, wherein discharged water is deposited in the bottom basin, and is recycled into the top basin through a water suction pump (2 liters/minute) so as to keep the water level in the basins stable; the P (LLA-CL)/SF spinning solution is placed in an upper injector, under the high pressure of 12kv, the solution jet velocity is 1.2mL/h, the distance is 8 cm-10 cm above a water vortex, and a lower roller rotates in water flow at a lower velocity to collect nano yarn; drying the collected membrane to obtain a P (LLA-CL)/SF two-dimensional nanofiber membrane;
s3: preparing a hyaluronic acid solution: preparing 100mL of 50mM MES buffer solution, adjusting the pH to 5, adding 1g of hyaluronic acid, adding 0.575g of EDC and 0.092g of NHS, and activating carboxyl for 30min to obtain a hyaluronic acid solution;
s4: immersing the P (LLA-CL)/SF two-dimensional nanofiber membrane into a hyaluronic acid solution, taking out after 6h, putting the membrane in a refrigerator at minus 80 ℃ overnight, and freeze-drying to obtain the two-dimensional nanofiber membrane of the cross-linked hyaluronic acid;
s5: placing a two-dimensional nanofiber membrane of cross-linked hyaluronic acid in 0.1M NaBH4Foaming in the aqueous solution, taking the scaffold out of foaming liquid by controlling the foaming time to be 5min, and then cleaning in deionized water for three times to obtain a three-dimensional nanofiber scaffold;
s6: the three-dimensional nanofiber scaffold is placed in a refrigerator at the temperature of-80 ℃ overnight, and then freeze-dried to obtain the articular cartilage scaffold with a multilayer three-dimensional structure.
The prepared articular cartilage scaffold is detected, and the corresponding detection results are shown in detail in figures 1-4.
Through detection, the thickness of the articular cartilage scaffold prepared in the embodiment is 3mm, the fiber diameter is 800-1000 nm, the pore diameter is 20-30 μm, and the interlayer spacing of the multilayer structure is 40-60 μm.
Planting new zealand white rabbit ear chondrocytes on the prepared articular cartilage support, and culturing at 37 ℃ under the condition of 5% carbon dioxide; after three days of culture, the cell activity is measured by a CCK-8 method; after three days of culture, the cell activity of the chondrocytes is 93 percent through detection; DAPI staining is carried out on the cultured chondrocytes for three days, then the cartilage support is cut, a confocal fluorescence microscope is adopted to shoot the section of the articular cartilage support, and the migration distance of the cells to the articular cartilage support is measured; the average distance of cell migration into the articular cartilage scaffold was determined to be 150 μm.
Example 2
This example provides a method for preparing an articular cartilage scaffold, in which the foaming time in step S5 is 20min, and the rest is the same as in example 1.
And detecting the prepared articular cartilage scaffold.
Through detection, the thickness of the articular cartilage scaffold prepared in the embodiment is 5mm, the fiber diameter is 800-1000 nm, the pore diameter is 20-30 μm, and the interlayer spacing of the multilayer structure is 60-80 μm.
Planting new zealand white rabbit ear chondrocytes on the prepared articular cartilage support, and culturing at 37 ℃ under the condition of 5% carbon dioxide; after three days of culture, the cell activity is measured by a CCK-8 method; after three days of culture, the cell activity of the chondrocytes is 94% by detection; DAPI staining is carried out on the cultured chondrocytes for three days, then the cartilage support is cut, a confocal fluorescence microscope is adopted to shoot the section of the articular cartilage support, and the migration distance of the cells to the articular cartilage support is measured; the average distance of cell migration into the articular cartilage scaffold was determined to be 200 μm.
Example 3
This example provides a method for preparing an articular cartilage scaffold, which includes the following steps:
s1: preparing a spinning solution: weighing 0.8g of Polycaprolactone (PCL) with molecular weight of eight ten thousand and 0.2g of Silk Fibroin (SF), dissolving in 10mL of Hexafluoroisopropanol (HFIP), and preparing into a PCL/SF spinning solution with mass concentration of 10%;
s2: preparing a two-dimensional nanofiber membrane: preparing a PCL/SF two-dimensional nanofiber membrane by a roller receiving electrostatic spinning method, wherein the device consists of an electrostatic spinning system and a roller receiver; placing the PCL/SF spinning solution in an injector, under the high pressure of 12kv, enabling the solution jet velocity to be 1.2mL/h, the distance to a roller to be 8-10 cm, enabling the roller rotation speed to be 200r/min, and drying the collected membrane in vacuum to obtain the PCL/SF two-dimensional nanofiber membrane;
s3: preparing a hyaluronic acid solution: preparing 100mL of 50mM MES buffer solution, adjusting the pH to 5, adding 1g of hyaluronic acid, adding 0.575g of EDC and 0.092g of NHS, and activating carboxyl for 30min to obtain a hyaluronic acid solution;
s4: immersing the PCL/SF two-dimensional nanofiber membrane into a hyaluronic acid solution, taking out after 6h, putting the PCL/SF two-dimensional nanofiber membrane into a refrigerator at the temperature of-80 ℃ overnight, and freeze-drying to obtain the two-dimensional nanofiber membrane of the cross-linked hyaluronic acid;
s5: placing a two-dimensional nanofiber membrane of cross-linked hyaluronic acid in 0.1M NaBH4Foaming in the aqueous solution, taking the scaffold out of foaming liquid by controlling the foaming time to be 5min, and then cleaning in deionized water for three times to obtain a three-dimensional nanofiber scaffold;
s6: the three-dimensional nanofiber scaffold is placed in a refrigerator at the temperature of-80 ℃ overnight, and then freeze-dried to obtain the articular cartilage scaffold with a multilayer three-dimensional structure.
And detecting the prepared articular cartilage scaffold.
Through detection, the thickness of the articular cartilage scaffold prepared in the embodiment is 3mm, the fiber diameter is 800-1000 nm, the pore diameter is 20-30 μm, and the interlayer spacing of the multilayer structure is 50-60 μm.
Planting new zealand white rabbit ear chondrocytes on the prepared articular cartilage support, and culturing at 37 ℃ under the condition of 5% carbon dioxide; after three days of culture, the cell activity is measured by a CCK-8 method; after three days of culture, the cell activity of the chondrocytes is 92% through detection; DAPI staining is carried out on the cultured chondrocytes for three days, then the cartilage support is cut, a confocal fluorescence microscope is adopted to shoot the section of the articular cartilage support, and the migration distance of the cells to the articular cartilage support is measured; the average distance of cell migration into the articular cartilage scaffold was determined to be 150 μm.
Example 4
This example provides a method for preparing an articular cartilage scaffold, in which the foaming time in step S5 is 20min, and the rest is the same as in example 3.
And detecting the prepared articular cartilage scaffold.
Through detection, the thickness of the articular cartilage scaffold prepared in the embodiment is 5mm, the fiber diameter is 800-1000 nm, the pore diameter is 20-30 μm, and the interlayer spacing of the multilayer structure is 60-80 μm.
Planting new zealand white rabbit ear chondrocytes on the prepared articular cartilage support, and culturing at 37 ℃ under the condition of 5% carbon dioxide; after three days of culture, the cell activity is measured by a CCK-8 method; after three days of culture, the cell activity of the chondrocytes is 93 percent through detection; DAPI staining is carried out on the cultured chondrocytes for three days, then the cartilage support is cut, a confocal fluorescence microscope is adopted to shoot the section of the articular cartilage support, and the migration distance of the cells to the articular cartilage support is measured; the average distance of cell migration into the articular cartilage scaffold was determined to be 200 μm.
Comparative example 1
This example provides a method for preparing an articular cartilage scaffold, which includes the following steps:
s1: preparing a spinning solution: weighing 0.8g of polylactic acid-polycaprolactone (P (LLA-CL)) with molecular weight of eight ten thousand and 0.2g of Silk Fibroin (SF) and dissolving in 10mL of Hexafluoroisopropanol (HFIP) to prepare P (LLA-CL)/SF spinning solution with mass concentration of 10%;
s2: preparing a two-dimensional nanofiber membrane: preparing a P (LLA-CL)/SF two-dimensional nanofiber membrane by dynamic water flow electrostatic spinning, and spinning by adopting a water circulation system and a device; the device consists of two basins, namely a top basin and a bottom basin, wherein discharged water is deposited in the bottom basin, and is recycled into the top basin through a water suction pump (2 liters/minute) so as to keep the water level in the basins stable; the P (LLA-CL)/SF spinning solution is placed in an upper injector, under the high pressure of 12kv, the solution jet velocity is 1.2mL/h, the distance is 8 cm-10 cm above a water vortex, and a lower roller rotates in water flow at a lower velocity to collect nano yarn; drying the collected membrane to obtain a P (LLA-CL)/SF two-dimensional nanofiber membrane;
s3: placing the P (LLA-CL)/SF two-dimensional nanofiber membrane in 0.1M NaBH4Foaming in water solution, taking out the scaffold from foaming solution by controlling foaming time to be 5min, and cleaning in deionized water for three times to obtain sodiumA rice fiber scaffold;
s4: the nanofiber scaffolds were placed in a refrigerator at-80 ℃ overnight and then freeze-dried to obtain articular cartilage scaffolds having a porous structure.
And detecting the prepared articular cartilage scaffold.
Through detection, the thickness of the articular cartilage scaffold prepared in the embodiment is 3mm, the fiber diameter is 800-900 nm, the pore diameter is 20-30 μm, and the interlayer spacing of the multilayer structure is 40-50 μm.
Planting new zealand white rabbit ear chondrocytes on the prepared articular cartilage support, and culturing at 37 ℃ under the condition of 5% carbon dioxide; after three days of culture, the cell activity is measured by a CCK-8 method; the cell activity of the chondrocytes is 88% after three days of culture; DAPI staining is carried out on the cultured chondrocytes for three days, then the cartilage support is cut, a confocal fluorescence microscope is adopted to shoot the section of the articular cartilage support, and the migration distance of the cells to the articular cartilage support is measured; the average distance of cell migration into the articular cartilage scaffold was determined to be 100 μm.
The above examples 1 and 2, and 3 and 4, differ only in the foaming time; as can be seen from a comparison of the thicknesses of the articular cartilage scaffolds obtained in examples 1 and 2, and examples 3 and 4, the thickness of the articular cartilage scaffold prepared was increased as the foaming time was increased under the same other experimental conditions.
Comparative example 1 is different from example 1 in that hyaluronic acid is not chemically cross-linked on the prepared two-dimensional nanofiber membrane before the foaming step in comparative example 1; as can be seen from fig. 4, under the same other reaction conditions, the compression performance of the articular cartilage scaffold prepared by chemically crosslinking hyaluronic acid on the two-dimensional nanofiber membrane in example 1 is obviously improved compared with that of the uncrosslinked hyaluronic acid in comparative example 1; therefore, the hyaluronic acid is crosslinked before foaming, and due to the hydrogen bonds formed between hyaluronic acid fiber networks and the new amido bonds formed between fibroin amino and hyaluronic acid carboxyl, the nanofiber membrane of the crosslinked hyaluronic acid can better simulate the articular cartilage extracellular matrix in chemical composition, is favorable for enhancing the mechanical property of the articular cartilage extracellular matrix, improves the compression property of the articular cartilage scaffold, and is favorable for the permeation of cells into the scaffold.
Although the present disclosure has been described above, the scope of the present disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure, and such changes and modifications will fall within the scope of the present invention.
Claims (10)
1. The preparation method of the articular cartilage scaffold is characterized by comprising the following steps:
s1: dissolving degradable polymer and natural high molecular material in a solvent to prepare spinning solution;
s2: performing electrostatic spinning on the spinning solution through electrostatic spinning equipment to obtain a two-dimensional nanofiber membrane;
s3: preparing a hyaluronic acid solution;
s4: dipping the two-dimensional nanofiber membrane in the hyaluronic acid solution, and freeze-drying to obtain a two-dimensional nanofiber membrane of cross-linked hyaluronic acid;
s5: placing the two-dimensional nanofiber membrane of the cross-linked hyaluronic acid in a foaming agent, and foaming to obtain a three-dimensional nanofiber scaffold;
s6: and drying the three-dimensional nanofiber scaffold to obtain the articular cartilage scaffold with a multilayer three-dimensional structure.
2. The method of preparing an articular cartilage scaffold according to claim 1, wherein the preparing the hyaluronic acid solution comprises:
s31: preparing MES buffer solution;
s32: adding hyaluronic acid into the MES buffer solution to obtain a MES buffer solution of the hyaluronic acid;
s33: and adding EDC/NHS into the MES buffer solution of the hyaluronic acid, and carrying out carboxyl activation reaction to obtain the hyaluronic acid solution.
3. The method for preparing an articular cartilage scaffold according to claim 1 or 2, characterized in that the foaming agent is an aqueous solution of sodium borohydride.
4. The method for preparing an articular cartilage scaffold according to claim 3, wherein the foaming time is 5 to 30 min.
5. The method according to claim 4, wherein the spinning solution of step S1 contains the degradable polymer and the natural polymer material at a total concentration of 8-15% by mass.
6. The method for preparing an articular cartilage scaffold according to claim 5, wherein the mass ratio of the degradable polymer to the natural high molecular material is 4: 1.
7. the method for preparing an articular cartilage scaffold according to claim 6, wherein the degradable polymer is one selected from the group consisting of polylactic acid-caprolactone copolymer and polycaprolactone.
8. The method for preparing an articular cartilage scaffold according to claim 6, wherein the natural polymer material is selected from one of silk fibroin and collagen.
9. The method for preparing an articular cartilage scaffold according to claim 6, wherein the solvent in the step S1 is one selected from the group consisting of hexafluoroisopropanol and dichloromethane.
10. An articular cartilage scaffold characterized by being produced by the method for producing an articular cartilage scaffold according to any one of claims 1 to 9.
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| CN115873143A (en) * | 2022-12-08 | 2023-03-31 | 诺一迈尔(山东)医学科技有限公司 | Core-shell bilayer structure microsphere and preparation method and application thereof |
| CN115873143B (en) * | 2022-12-08 | 2024-03-22 | 诺一迈尔(山东)医学科技有限公司 | Microsphere with core-shell double-layer structure and preparation method and application thereof |
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Application publication date: 20211207 |