CN107604468B - Processing method and application of micron-sized biological material fiber - Google Patents
Processing method and application of micron-sized biological material fiber Download PDFInfo
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- CN107604468B CN107604468B CN201710918764.2A CN201710918764A CN107604468B CN 107604468 B CN107604468 B CN 107604468B CN 201710918764 A CN201710918764 A CN 201710918764A CN 107604468 B CN107604468 B CN 107604468B
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Abstract
The invention discloses a processing method of micron-sized biomaterial fiber and application thereof, comprising the following steps of 1) fiber forming: firstly, a pseudoplastic material and a biological material to be processed are used for manufacturing a ring with a core-sheath structure, wherein the core is a processed biological material solution, the sheath is a pseudoplastic material, the ring is softened by heating, and the ring is extruded and stretched to enlarge the radius of the ring; repeating the steps, and folding for N times; folding to the required size, and cooling and solidifying the ring; 2) fiber treatment: cutting the required length from the ring, and dissolving the thermoplastic plastic by using an organic solvent to obtain the remaining material, namely the biological material; putting the biological material into a methanol solvent, adding a cross-linking agent, and fully mixing to cross-link the biological material fibers; washing the crosslinked biomaterial fiber with an organic solvent to reduce the concentration of the crosslinking agent to a specified concentration; 3) and (4) storing the fibers. The invention can be customized and produced according to the self condition and cell types of patients; and the production cost is low, the efficiency is high, and the large-scale production of clinical requirements can be met.
Description
Technical Field
The invention relates to a processing method and application of micron-sized biological material fibers, belonging to the field of processing of biological material fiber yarns.
Background
Cell therapy, regenerative medicine or tissue engineering generally utilizes biomaterial scaffolds as carriers to implant and control cell growth. The micron-sized filamentous biomaterial scaffold, especially the micron fiber prepared from natural protein or polysaccharide extracted from biological tissues, can provide biochemical signals and biomechanical stimulation for cells simulating three-dimensional environment of human body, and simultaneously provide sufficient nutrition supply and growth space for the cells, so the protein or polysaccharide micron fiber has wide application prospect in the fields of cell therapy, regenerative medicine and tissue engineering. However, the existing micron fiber processing method is too long to meet the clinical application requirements, and more importantly, the existing processing method has strict requirements on the physical and chemical properties of materials and is not suitable for natural materials extracted from biological tissues, such as protein and polysaccharide.
Currently common processing methods include electrospinning, melt spinning, wet spinning, and 3D printing. Electrostatic spinning: static electricity is formed on a drop of liquid after a sufficiently high voltage is applied to the drop. The repulsive force between the charges counteracts the surface tension of the liquid, causing the droplets to elongate and form fibers. Melt spinning: and (3) passing the high molecular polymer melt through the small holes to form melt trickle, and solidifying the melt trickle into fibers. Such as the production of cotton candy. Wet spinning: the high molecular polymer is dissolved in a solvent, and the solution is sprayed into a coagulating bath through a spray head, so that the high molecular polymer is separated out to form fibers. 3D printing: the high molecular polymer is dissolved in a solvent, and is made into ink by using an additive to print a strip-shaped structure.
The processing method has the following defects: electrostatic spinning: 1. only materials with a certain conductivity can be processed, but the protein solution is not conductive. 2. The spinning speed is too slow, and the spinning method is only suitable for a small amount of use in a laboratory and cannot be used for clinical scale production. 3. Collecting the fibers can cause the fibers to adhere to each other and prevent uniform three-dimensional cell encapsulation. Melt spinning: 1. only materials with a specific melting point can be processed, but such high temperatures can destroy the chemical structure of the protein. 2. The resulting fiber diameter is typically above 20 microns. 3. The accuracy is poor. 4. Cells cannot be encapsulated uniformly. Wet spinning: 1. only materials with specific solubility and precipitation properties can be processed. 2. The resulting fiber diameter is typically over 50 microns, much larger than the cell size. 3. The production efficiency is low. 3D printing: 1. the "ink" is required to have a specific viscosity and melting point to be printable. 2. The fiber diameter produced is limited by the jet size and "drop" spread, typically above 100 microns, much larger than the cell size. 3. The cost of equipment required to print fine fibers rises dramatically and the time taken is exponentially increased.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a processing method of micron-sized biomaterial fiber and application thereof, which solve the problem that most of technologies cannot produce natural biomaterial fiber filaments such as protein, polysaccharide and the like.
In order to achieve the purpose, the processing method of the micron-sized biomaterial fiber adopted by the invention specifically comprises the following steps:
1) fiber forming:
firstly, manufacturing a ring of a core-sheath structure, wherein the core is made of processed biological materials, and the sheath is made of pseudoplastic materials;
secondly, heating to soften the ring, and extruding the stretching ring to enlarge the radius of the stretching ring;
step three, folding the ring in half, overlapping the two halves together, continuously extruding and stretching the ring, and simultaneously enlarging the radius;
repeating the above steps, folding for N times to increase the biological material in the hollow rod 2NMultiple, 2 of the original diameter reduced in diameter0.5NOne-fourth;
fourthly, after the ring is folded to the required size, the ring is placed in cold water or air to be cooled and solidified;
2) fiber treatment:
firstly, cutting a required length from a ring, and dissolving a pseudoplastic material by using an organic solvent to obtain a remaining material which is a biological material;
secondly, putting the biological material into a methanol solvent, adding a cross-linking agent, and fully mixing to cross-link the biological material fibers;
thirdly, fully cleaning the crosslinked biomaterial fiber by using an organic solvent to reduce the concentration of the crosslinking agent to a specified concentration;
3) and (4) storing the fibers.
As a refinement, the biological material is protein or polysaccharide.
As an improvement, the fiber storage in the step 3) specifically comprises the steps of drying all organic solvents by high-pressure vacuum, and directly storing the organic solvents in an aseptic mode, or replacing the organic solvents with water by dialysis, and freeze-drying the organic solvents by liquid nitrogen, and then storing the organic solvents in an aseptic mode.
As an improvement, in the fiber forming in the step 1), the ring is folded in half to form a 8 shape in the fourth step, and then the two halves of the folded ring are overlapped together to continuously extrude and stretch the ring.
Micron-sized biomaterial fibers produced by any of the above processing methods.
The use of the process for the production of gelatin fibres.
An application of gelatin fiber in articular cartilage injury repairing operation is provided.
Compared with the prior art, the invention has the beneficial effects that:
1) the invention can produce gram micrometer to nanometer fiber only in about 10min, which can meet the requirement of clinical cell therapy and tissue regeneration for scaffold material.
2) By adopting a folding and stretching process similar to that of a stretched surface, the fibers produced by the invention are aligned in parallel, the growth of a plurality of cells and the regeneration of tissues, such as myocardial repair, ligament repair and meniscus repair, must require materials to provide parallel topological structures, and the invention can effectively meet the strict requirements.
3) The invention can use the protein corresponding to the tissue which is pertinently needed to be repaired to carry out customized production according to the self condition and the cell type of the patient.
4) The invention has low production cost and high efficiency, and can meet the large-scale production of clinical requirements.
5) The invention solves the problem that most fiber processing technologies cannot be compatible with cell culture in the production process.
Drawings
FIG. 1 is a schematic view of the process of the present invention for dissolving the residual fibers of the plastic in the ring;
FIG. 2 is a schematic representation of the ring of the present invention being stretched in pairs to obtain fibers of different diameters;
FIG. 3 shows the results of in vitro culture of bone marrow stem cells at a density of 5 million/cc in 5 μm diameter fibers in example 1;
FIG. 4 shows the result of the instant PCR assay for RNA in example 1;
FIG. 5 is the mechanical results of in vitro culture at a density of 1 million/cc for 6 weeks in example 1;
FIG. 6 shows the three-dimensional scanning results of confocal fluorescence microscope after 1 week of in vitro stem cell culture (left) and 1 week of in vitro ligament progenitor cell culture (right) in example 2.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood, however, that the description herein of specific embodiments is only intended to illustrate the invention and not to limit the scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the terms used herein in the specification of the present invention are for the purpose of describing particular embodiments only and are not intended to limit the present invention.
A processing method of micron-sized biomaterial fiber specifically comprises the following steps:
1) fiber forming:
firstly, manufacturing a ring of a core-sheath structure, wherein the core is made of processed biological materials, and the sheath is made of pseudoplastic materials;
secondly, heating to soften the ring, and extruding the stretching ring to enlarge the radius of the stretching ring;
step three, folding the ring in half, overlapping the two halves together, continuously extruding and stretching the ring, and simultaneously enlarging the radius;
repeating the above steps, folding for N times to increase the biological material in the hollow rod 2NMultiple, reduced diameter to the original diameter2 of (2)0.5NOne-fourth;
fourthly, after the ring is folded to the required size, the ring is placed in cold water or air to be cooled and solidified;
2) fiber treatment:
firstly, cutting a required length from a ring, and dissolving a pseudoplastic material by using an organic solvent to obtain a remaining material which is a biological material;
secondly, putting the biological material into a methanol solvent, adding a cross-linking agent, and fully mixing to cross-link the biological material fibers;
thirdly, fully cleaning the crosslinked biomaterial fiber by using an organic solvent to reduce the concentration of the crosslinking agent to a specified concentration;
3) and (4) storing the fibers.
As a modification of the examples, the biological material is a protein or a polysaccharide. The method for processing the fiber is not limited to natural materials such as protein, polysaccharide and the like, but also can be expanded to artificially synthesized high molecular polymers.
As an improvement of the embodiment, the fiber storage in the step 3) specifically comprises the steps of drying all organic solvents by high-pressure vacuum, directly and aseptically storing, or replacing the organic solvents by water by dialysis, and aseptically storing after freeze-drying by liquid nitrogen.
As a modification of the embodiment, in the fiber forming of the step 1), the ring is folded in half to form a 8-shape in the fourth step, and then the two halves of the folded ring are overlapped together and the ring is continuously pressed and stretched. Of course, when the stretching ring is folded in half, the stretching ring is not limited to stretching a circular ring, and any shape, such as a rectangle, a square, a prism and the like, can play a similar role; the folding can be directly done by twisting, twisting three times, folding four times, etc. The number of folds and stretches was varied and fibers with diameters from 500 nm to 100 μm were obtained, as shown in FIG. 2.
Micron-sized biomaterial fibers produced by any of the above processing methods.
The use of the process for the production of gelatin fibres.
An application of gelatin fiber in articular cartilage injury repairing operation is provided.
Example 1
A processing method of gelatin fiber for articular cartilage damage repair surgery specifically comprises the following steps:
1) fiber forming:
making rings with core-sheath structure, wherein the core is 50% gelatin (cheap and equivalent collagen substitute) by weight, and the sheath is polycaprolactone;
heating the ring in oil bath with oil temperature of 70 deg.C until the ring is completely plasticized and becomes transparent, twisting, folding and stretching for 20 times, and cooling in water for solidification;
2) fiber treatment:
placing the ring in acetone at 30 deg.C for 12 hr to dissolve polycaprolactone to obtain fiber, as shown in FIG. 1;
replacing the cleaned fiber with new acetone at least five times, and maintaining the temperature of acetone at 30 deg.C for not less than 8 hr each time;
replacing the acetone with 2-propanol and shaking the vessel for at least 15 minutes or until the vessel is full of fibers;
replacing 2-propanol with methanol, and shaking the container for 3 minutes to make methanol fully permeate into gaps among fibers;
adding 2% by volume of methacrylic anhydride and 0.2% by weight of hydroquinone or glutaraldehyde (keeping the concentration of glutaraldehyde in the added solution at 0.1% to 2%), and mixing thoroughly for at least 15 minutes;
washing the fibers twice with methanol for at least 5 minutes each time;
washing the fibers with 2-propanol five times for at least 5 minutes each time;
3) fiber storage
Placing in high pressure vacuum for at least 24 hr, pumping out 2-propanol, keeping the whole process in shade, sterilizing, and storing the fiber in-20 deg.C refrigerator.
4) Cell encapsulation
Fully mixing the fibers and normal saline according to the weight ratio of 1:8 to form jelly, and placing the jelly at a shading position of 4 ℃ for 30 minutes to fill the gaps among the fibers with water;
the photoinitiator LAP (2,4, 6-trimethylbenzonyl) phosphate with a concentration of 0.5% was prepared with physiological saline.
The fiber dope was mixed well with the photoinitiator solution in a weight ratio of 1: 1.
Bone marrow Stem Cells (meschymal Stem Cells (MSCs)) were mixed well with the jelly at a density of 2 million/cc (measured according to the patient's actual condition) and stirred.
Irradiating with ultraviolet rays at 365nm and 4mWcm-2And setting for 4 minutes.
And (3) placing the cell product in a chondrocyte culture solution for culture, induction and differentiation, or carrying out autologous stem cell cartilage transplantation operation.
The following are the results of in vitro culture of bone marrow stem cells at a density of 5 million/cubic centimeter in 5 micron diameter fibers:
as in fig. 3, tissue staining. The left panel shows the day 0 staining results, with fibers as bands and cells as dots. The right panel shows the result of staining on day 21, degradation of the gelatin fibers and formation of new tissue.
FIG. 4, RNA immediate polymerase chain reaction assay, comprising 5 genes: SOX9, Aggrecan, type 1 collagen, type 10 collagen, type 2 collagen. The major component of cartilage tissue, collagen type 2, is increased by nearly a million fold.
FIG. 5 shows that the mechanical properties of the culture can reach 60% of those of autologous cartilage tissue after in vitro culture for 6 weeks at a density of 1 million/cubic centimeter.
Example 2
A processing method of parallel gelatin fibers for ligament injury repair surgery specifically comprises the following steps:
1) fiber forming:
making rings with core-sheath structure, wherein the core is 50% gelatin (cheap and equivalent collagen substitute) by weight, and the sheath is polycaprolactone;
heating the ring in an oil bath with oil temperature of 50 deg.C until the ring is completely plasticized and becomes transparent, twisting, folding and stretching for 20 times, and cooling in water for solidification;
2) fiber treatment:
evenly dividing the ring into equal parts, and clamping or fixing two ends of each equal part;
placing each section in acetone at 30 ℃ for 12 hours, and dissolving polycaprolactone to obtain mutually parallel fibers;
keeping the fibers parallel, replacing the cleaned fibers with new acetone at least five times, and keeping the temperature of the acetone at 30 ℃ for not less than 8 hours each time;
replacing acetone with 2-propanol and soaking the fibers for at least 15 minutes;
replacing 2-propanol with methanol to make methanol fully penetrate into gaps between fibers;
adding 2% by volume of methacrylic anhydride and 0.2% by weight of hydroquinone or glutaraldehyde (keeping the concentration of the glutaraldehyde in the added solution at 0.1-2%) and stirring thoroughly for at least 15 minutes;
washing the fibers twice with methanol for at least 5 minutes each time;
washing the fibers with 2-propanol five times for at least 5 minutes each time;
3) fiber storage
Placing in high pressure vacuum for at least 24 hr, pumping out 2-propanol, keeping the whole process in shade, sterilizing, and storing the fiber in-20 deg.C refrigerator.
4) Cell encapsulation
Keeping the fibers parallel, fully mixing the fibers and normal saline according to the weight ratio of 1:8 to form jelly, and standing at a shading position at 4 ℃ for 30 minutes to fill the gaps among the fibers with water;
the photoinitiator LAP (2,4, 6-trimethylbenzonyl) phosphate with a concentration of 0.5% was prepared with physiological saline.
Keeping the fibers parallel, and fully mixing the fiber jelly and the photoinitiator solution according to the weight ratio of 1: 1.
Bone Marrow Stem Cells (MSCs) or ligament progenitor Cells (TPCs) are repeatedly washed through parallel fibers at a density of 2 million/cubic centimeter (or measured according to patient conditions) to allow the Cells to fully enter the fibers.
Irradiating with ultraviolet rays at 365nm and 4mWcm-2And setting for 4 minutes.
FIG. 6 (left), stem cells cultured in vitro for 1 week, confocal fluorescence microscopy three-dimensional scan results. With blue nuclei, green cytoskeleton.
FIG. 6 (right), the ligament progenitor cells cultured in vitro for 1 week, and the results of confocal fluorescence microscope three-dimensional scanning. With blue nuclei, green cytoskeleton.
Example 3
Hyaluronic acid fibers were prepared by following the procedure of example 1 using a 5% hyaluronic acid (or hyaluronic acid) aqueous solution as a core preparation ring.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (6)
1. The processing method of the micron-sized biomaterial fiber is characterized by comprising the following steps of:
1) fiber forming:
firstly, preparing a ring of a core-sheath structure, wherein the core is a processed biological material, the sheath is a pseudoplastic material, and the biological material is protein or polysaccharide;
secondly, heating to soften the ring, and extruding the stretching ring to enlarge the radius of the stretching ring;
step three, folding the ring in half, overlapping the two halves together, continuously extruding and stretching the ring, and simultaneously enlarging the radius;
repeating the above steps, folding for N times to increase the biological material in the hollow rod to 2 timesNMultiple, 2 of the original diameter reduced in diameter0.5NOne-fourth;
fourthly, after the ring is folded to the required size, the ring is placed in cold water or air to be cooled and solidified;
2) fiber treatment:
firstly, cutting a required length from a ring, and dissolving a pseudoplastic material by using an organic solvent to obtain a remaining material which is a biological material;
secondly, putting the biological material into a methanol solvent, adding a cross-linking agent, and fully mixing to cross-link the biological material fibers;
thirdly, fully cleaning the crosslinked biomaterial fiber by using an organic solvent to reduce the concentration of the crosslinking agent to a specified concentration;
3) and (4) storing the fibers.
2. The method for processing micron-sized biomaterial fiber as claimed in claim 1, wherein the fiber storage in step 3) specifically comprises using high-pressure vacuum to evacuate all organic solvents, and directly storing the dried micron-sized biomaterial fiber in an aseptic manner, or using dialysis to replace the organic solvents with water, and using liquid nitrogen to freeze and store the dried micron-sized biomaterial fiber in an aseptic manner.
3. The method as claimed in claim 1, wherein the step 1) is performed by folding the ring in half into 8 shape, and then overlapping the two halves of the folded ring, and further pressing and stretching the ring.
4. Micron-sized biomaterial fiber, characterized in that it is obtained by the process according to any one of claims 1 to 3.
5. Use of a process according to any one of claims 1 to 3 on gelatin fibres.
6. Use of the gelatin fiber prepared in claim 5 in articular cartilage damage repair surgery.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101525782A (en) * | 2008-03-04 | 2009-09-09 | 东丽纤维研究所（中国）有限公司 | Short fiber of polyethylene terephthalate and method for producing same |
| CN102166372A (en) * | 2011-02-14 | 2011-08-31 | 东南大学 | Manufacturing method of composite nanofiber scaffold for promoting repair of bone defect |
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| US20040067585A1 (en) * | 2002-10-07 | 2004-04-08 | Yu-Chi Wang | Cell cultivation surface and method of making the same |
| TWI374037B (en) * | 2008-08-21 | 2012-10-11 | Univ Taipei Medical | Bio-acceptable conduits and method providing the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101525782A (en) * | 2008-03-04 | 2009-09-09 | 东丽纤维研究所（中国）有限公司 | Short fiber of polyethylene terephthalate and method for producing same |
| CN102166372A (en) * | 2011-02-14 | 2011-08-31 | 东南大学 | Manufacturing method of composite nanofiber scaffold for promoting repair of bone defect |
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