Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide an ordered collagen scaffold material and a preparation method and application thereof. The product prepared by the preparation method has an orderly parallel arrangement collagen fiber structure, reserves the neuron attaching capability and the nerve fiber extending capability of collagen, and can remove residues of organic matters and reagents in the product so as to effectively regenerate nerve tissues after spinal cord injury is induced.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
In a first aspect, the present invention provides a method for preparing an ordered collagen scaffold material, the method comprising:
(1) Subjecting mammalian fascia tissue to a combination of electrical pulse stimulation and ultrasonic stimulation;
(2) Soaking the treated product in alkali liquor;
(3) Soaking the product subjected to alkali liquor treatment in an organic solution for preliminary degreasing treatment, and then performing degreasing treatment again by using supercritical carbon dioxide;
(4) Crosslinking the degreased product by using a chemical crosslinking agent;
(5) Fixing and forming the crosslinked product;
(6) And freeze-drying the fixed and molded product to obtain the ordered collagen scaffold material.
The invention creatively develops a collagen scaffold material for spinal cord injury repair and a preparation method thereof, wherein the method uses an ultrasonic and electric pulse dual physical field to pretreat tissues, and the two stimulation treatments are synergistic, so that the efficiency of removing cells is obviously improved compared with single ultrasonic stimulation or single electric pulse stimulation treatment, and the spinal cord repair effect is obviously improved; the combination mode does not need to add a traditional decellularizing reagent, so that the risks of solvent residue and collagen denaturation in the treatment process are greatly reduced, the decellularizing time is shorter, and the effect is better; meanwhile, the combination mode does not need to add enzymes for treatment, so that the problem that the collagen structure is easy to damage due to the use of enzymes, so that the attaching and extending efficiency of nerve cells is reduced is avoided, the decellularizing time is shorter, and the effect is better.
The method adopts the mode of combining the organic solvent with the supercritical carbon dioxide to realize efficient degreasing, so that not only can the lipid in the fascia tissue be removed more effectively, but also the risk of solvent residue is further reduced, the treatment process is mild, and the nerve repair efficiency of the collagen scaffold is not affected.
The method is also introduced with fixation molding treatment and freeze-drying treatment on the basis of chemical crosslinking treatment, so that the in-vivo stability of the material can be further enhanced, the structural order is realized, and the collagen content of the product and the attaching growth effect of nerve cells are improved.
In conclusion, the preparation method of the ordered collagen scaffold material greatly improves the material preparation efficiency, improves the collagen content of the material and the attaching growth effect of nerve cells and improves the spinal cord injury repair effect through the effective coordination of the steps.
Preferably, the combined treatment of step (1) is performed alternately or sequentially with electrical pulse stimulation and ultrasonic stimulation 1-5 times, for example 1 time, 2 times, 3 times, 4 times, 5 times.
The expression "alternately or sequentially carrying out 1-5 times" means that the electric pulse stimulation and the ultrasonic stimulation are alternately carried out 1-5 times respectively, or the electric pulse stimulation is carried out 1-5 times before the ultrasonic stimulation is carried out 1-5 times, or the ultrasonic stimulation is carried out 1-5 times before the electric pulse stimulation is carried out 1-5 times.
The electrical pulse stimulation uses electrodes to treat fascia tissue, the stimulation voltage is set to 10-100V (e.g., 10V, 20V, 30V, 40V, 50V, 60V, 70V, 80V, 90V, 100V, etc.), the pulse length is 10-250 ms (e.g., 20 ms, 40 ms, 50 ms, 70 ms, 80 ms, 100 ms, 120 ms, 150 ms, 170 ms, 180 ms, 200 ms, 250 ms, etc.), and the pulse interval is 1-7 s (e.g., 1 s, 2 s, 3 s, 4 s, 5 s, 6 s, 7 s, etc.), 10-100 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, etc.) pulse stimulations are performed each time.
The power of the ultrasonic stimulation is 50-200W (e.g., 50W, 70W, 80W, 100W, 120W, 140W, 150W, 160W, 170W, 180W, 200W, etc.), and the time is 1-10 min (e.g., 1 min, 2 min, 3 min, 4 min, 5min, 6 min, 7 min, 8 min, 9 min, 10min, etc.).
Preferably, the lye of step (2) is an aqueous NaOH solution of 0.5-3M (e.g., 0.5M, 0.7M, 0.8M, 1.0M, 1.3M, 1.5M, 2.0M, 2.5M, 3.0M, etc.), the soaking time is 0.5-3 h (e.g., 0.5 h, 0.7 h, 0.8 h, 0.9 h, 1.0 h, 1.2 h, 1.4 h, 1.5 h, 2h, 3 h, etc.), the soaking temperature is 2-10 ℃ (e.g., 2 ℃,3 ℃, 4 ℃,5 ℃,6 ℃,7 ℃, 8 ℃,10 ℃ etc.).
And cleaning the product after the soaking treatment.
Preferably, the organic solution of step (3) comprises a combination of ethanol and ethyl acetate in a volume ratio of ethanol to ethyl acetate of 1:1 to 1:4, e.g. 1:1, 1:2, 1:3, 1:4, etc.
The soaking time in the step (3) is 15-30 h (such as 15 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 24h, 25 h, 30 h, etc.), and the soaking temperature is 20-30 ℃ (such as 20 ℃, 22 ℃,24 ℃, 26 ℃, 28 ℃,30 ℃, etc.); and cleaning the product after the soaking treatment.
The organic solvent selected for degreasing in the preparation method of the ordered collagen scaffold material is more preferably a combination of ethanol and ethyl acetate, and compared with other degreasing solvents such as acetone, diethyl ether or other combination modes, the combination of ethanol and ethyl acetate has better degreasing effect, the fat content in the product is lower, and the spinal cord injury repairing effect is better.
Preferably, in the degreasing treatment of the supercritical carbon dioxide in the step (3), the pressure is 5-40 MPa (for example, 5 MPa, 10 MPa, 15 MPa, 20 MPa, 25 MPa, 30 MPa, 35 MPa, 40 MPa, etc.), the temperature of the supercritical carbon dioxide is 25-55 ℃ (for example, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃ etc.), and the flow rate of the supercritical carbon dioxide is 0.5-12L/min (for example, 1L/min, 3L/min, 4L/min, 6L/min, 8L/min, 10L/min, 12L/min, etc.), and the time is 20-40min (for example, 20min, 25min, 30min, 35min, 40min, etc.).
Preferably, the chemical cross-linking agent of step (4) comprises N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide; the crosslinking treatment is carried out at a temperature of 30 to 40 ℃ (e.g., 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 37 ℃, 39 ℃ and the like) for a time of 0.5 to 1.5 h (e.g., 0.5 h, 0.7 h, 0.8 h, 0.9 h, 1.0 h, 1.2 h, 1.4 h, 1.5 h and the like).
The product is also cleaned after the crosslinking treatment.
Preferably, the fixing and forming in the step (5) is performed by alternately soaking the product in ethanol solution and water; alternating 3-8 times (e.g., 3 times, 4 times, 5 times, 6 times, 7 times, 8 times), each soaking time is 3-8 min times (e.g., 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, etc.); wherein the ethanol concentration of the ethanol solution is 75-100% (e.g., 75%, 80%, 85%, 90%, 95%, 100%, etc.).
Preferably, the freeze-drying in step (6) is performed by pre-freezing, and the freeze-drying procedure is as follows: the first stage: -35 to-25 ℃,1-5 h; and a second stage: -25 to-15 ℃ and 0.5 to 1.5 h; and a third stage: -25 to-15 ℃ and 5 to 15 h; fourth stage: -15 to-5 ℃ and 0.5 to 1.5 h; fifth stage: -15 to-5 ℃ and 10 to 20 h; sixth stage: -5 ℃,1-5 h; seventh stage: 5-15 ℃ and 1-5 h; eighth stage: 10-20 ℃ and 1-5 h; ninth stage: 20-30 ℃ and 1-5 h; in the second stage, a vacuum is drawn.
Specific point values in the range from minus 35 ℃ to minus 25 ℃ can be selected from minus 35 ℃, minus 32 ℃, minus 30 ℃, minus 28 ℃, minus 27 ℃, minus 26 ℃, minus 25 ℃ and the like; specific point values in the "1-5 h" can be selected as 1 h, 2 h, 3 h, 4 h, 5 h, etc.; specific point values in the range from minus 25 ℃ to minus 15 ℃ can be selected from minus 25 ℃, minus 22 ℃, minus 20 ℃, minus 18 ℃, minus 17 ℃, minus 16 ℃, minus 15 ℃ and the like; specific point values in the "0.5-1.5 h" may be selected as 0.5 h, 0.7 h, 0.8 h, 1 h, 1.2 h, 1.5 h, etc.; specific point values in the "5-15 h" can be selected as 5 h, 8 h, 10 h, 12 h, 14 h, 15 h, etc.; specific point values in the range from minus 15 ℃ to minus 5 ℃ can be selected from minus 15 ℃, minus 12 ℃, minus 10 ℃, minus 8 ℃, minus 7 ℃, minus 6 ℃, minus 5 ℃ and the like; the specific point values in the "10-20 h" can be selected as 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, etc.; specific values in the range of-5 to 5 ℃ can be selected from-5 ℃,2 ℃ and 0 ℃,3 ℃,4 ℃ and 5 ℃ and the like; the specific point value in the' 5-15 ℃ can be selected as 5 ℃, 7 ℃, 8 ℃, 9 ℃, 10 ℃, 12 ℃, 15 ℃ and the like; the specific point value in the temperature of 10-20 ℃ can be 10 ℃, 12 ℃, 14 ℃, 15 ℃, 16 ℃, 18 ℃, 20 ℃ and the like; the specific point value in the temperature range of 20-30 ℃ can be selected as 20 ℃, 22 ℃, 24 ℃, 25 ℃, 26 ℃,28 ℃, 30 ℃ and the like; other specific point values in the numerical ranges are selectable, and will not be described in detail herein.
The preparation method of the ordered collagen scaffold material has the advantages that the preparation method of the ordered collagen scaffold material is characterized in that a freeze-drying treatment program is specially designed, nine stages are performed, each stage meets a specific temperature interval and a specific time interval, the structure of a final product is more ordered, and the stability of the material in the body and the spinal cord repairing effect are enhanced.
In a second aspect, the present invention provides an ordered collagen scaffold material prepared according to the preparation method of the first aspect.
In a third aspect, the present invention provides the use of an ordered collagen scaffold material according to the second aspect in a product for spinal cord injury repair.
In a fourth aspect, the present invention provides a product for spinal cord injury repair comprising the ordered collagen scaffold material of the second aspect and cetuximab supported on the ordered collagen scaffold material;
The product for repairing the spinal cord injury is prepared by a preparation method comprising the following steps:
The ordered collagen scaffold material is soaked in cetuximab solution and incubated at 0-30deg.C (e.g., 1 deg.C, 2 deg.C, 3 deg.C, 4 deg.C, 5 deg.C, 6 deg.C, 7 deg.C, 8 deg.C, 9 deg.C, 10 deg.C, 15 deg.C, 20 deg.C, 25 deg.C, 30 deg.C, etc.) for 20-60 min (e.g., 20min, 30 min, 40min, 50 min, 60min, etc.). Other specific point values in the numerical ranges are selectable, and will not be described in detail herein.
After spinal cord injury, glial cell activation and proliferation can occur, resulting in the formation of glial scars. It has been shown that reactive astrocytes present after spinal cord injury express a large amount of nerve regeneration inhibitory molecules such as Chondroitin Sulfate Proteoglycan (CSPGs). Chondroitin sulfate proteoglycans not only spatially affect axon regeneration in the form of scars, but also activate the intra-neuronal Protein Kinase C (PKC) signaling pathway which in turn activates Rho to inhibit nerve regeneration. The Epidermal Growth Factor Receptor (EGFR) activates Rho-Rac signaling upon activation to inhibit axon regeneration. The drug currently used in the clinic for EGFR antibodies is Cetuximab. The research results prove that in the spinal cord injury environment, the cetuximab can effectively reduce the glial scar in the injury area and inhibit the deposition of molecular chondroitin sulfate proteoglycan, thereby promoting nerve regeneration. Therefore, the ordered collagen is utilized to guide the ordered growth of nerve fibers, the compound medicament cetuximab further reduces scar generation, and the two cooperate to better reconstruct the damaged microenvironment and enhance nerve regeneration. The composite functional nerve regeneration collagen scaffold provides a new method for the application and treatment of clinical spinal cord injury repair.
Compared with the prior art, the invention has the following beneficial effects:
The ordered collagen scaffold material prepared by the preparation method has an ordered parallel arrangement collagen fiber structure, reserves the nerve cell attaching capability and the nerve fiber extending capability of collagen, and can remove residues of organic matters and reagents in the product so as to effectively regenerate nerve tissues after spinal cord injury is induced. The method comprises the steps of performing pretreatment on tissues by using an ultrasonic and electric pulse dual physical field, wherein the two stimulation treatments are synergistic, and compared with single ultrasonic stimulation or single electric pulse stimulation treatment, the efficiency of removing cells is obviously improved; the method has the advantages that the method adopts the combination of the organic solvent and the supercritical carbon dioxide to realize efficient degreasing, so that not only can the lipid in the fascia tissue be removed more effectively, but also the risk of solvent residue is further reduced, the treatment process is mild, and the nerve repair efficiency of the collagen scaffold is not affected; the method is characterized in that the method is also introduced with fixation forming treatment and freeze-drying treatment on the basis of chemical crosslinking treatment, so that the in-vivo stability of the material can be further enhanced, the structural order is realized, and the collagen content of the product and the attaching growth effect of nerve cells are improved.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
Example 1
The embodiment provides an ordered collagen scaffold material, and the preparation method thereof is as follows:
(1) Fresh fascia tissue was taken, physically shaved off muscle and adipose tissue, and soaked in deionized water at 4 ℃ for 2h, with water changed every half hour.
(2) After immersing the tissue in water at 4 ℃ for 15 minutes, the tissue is subjected to electric pulse stimulation by using an electrode, the stimulation voltage is set to 90V, the pulse length is 100ms, the pulse interval is 2s, and 50 pulse stimulations are performed.
(3) Immersing the treated tissue in water at 16 ℃ for ultrasonic stimulation with the power of 100W and the stimulation time of 5 min.
(4) The steps (2) and (3) are repeated 3 times.
(5) The treated tissue was soaked in 2M NaOH aqueous solution for 1h, then the alkali solution was removed and rinsed 10 times with deionized water 5min each.
(6) The treated tissue was soaked in a mixed solution of ethanol and ethyl acetate (volume ratio of 1:2) at 16 ℃ for 10h times, and then rinsed with deionized water for 5 times min each time.
(7) The treated tissue is treated by supercritical carbon dioxide under the pressure of 30 MPa and the temperature of 30 ℃, the flow rate of the carbon dioxide is 6L/min, and the time is 30 min.
(8) The treated tissue was treated with 1mg/ml EDC and 0.5mg/ml NHS solution for 1h, and the tissue was collected and washed 3 times with deionized water, 5min each.
(9) The treated tissue is soaked in 100% ethanol solution 5min, and then the solution is changed into deionized water 5min, so that the treatment is carried out repeatedly for 5 times. Then soaked in deionized water for 30min a.
(10) Performing freeze-drying treatment, pre-freezing at-70 ℃ for 4h, then putting into a freeze dryer for freeze-drying, and vacuumizing in the 2 nd stage, wherein the freeze-drying degree is as follows:
Example 2
The embodiment provides an ordered collagen scaffold material, and the preparation method thereof is as follows:
(1) Fresh fascia tissue was taken, physically shaved off muscle and adipose tissue, and soaked in deionized water at 4 ℃ for 2h, with water changed every half hour.
(2) After immersing the tissue in water at 4 ℃ for 15 minutes, the tissue is subjected to electric pulse stimulation by using an electrode, the stimulation voltage is set to 90V, the pulse length is 200ms, the pulse interval is 1s, and 100 pulse stimulation is performed.
(3) Immersing the treated tissue in water at 16 ℃ for ultrasonic stimulation with the power of 200W and the stimulation time of 3 min.
(4) The steps (2) and (3) are repeated 3 times.
(5) The treated tissue was soaked in 1M NaOH aqueous solution for 1.5 h times, then the alkaline solution was removed and rinsed 10 times with deionized water 5min times.
(6) The treated tissue was soaked in a mixed solution of ethanol and ethyl acetate (volume ratio of 1:3) at 16 ℃ for 15 h times, and then rinsed with deionized water 10 times, each time 5: 5 min.
(7) The treated tissue is treated by supercritical carbon dioxide under the pressure of 25 MPa and the temperature of 35 ℃, the flow rate of the carbon dioxide is 3L/min, and the time is 30 min.
(8) The treated tissue was treated with 1mg/ml EDC and 0.5mg/ml NHS solution at 16deg.C for 1 h, and the tissue was collected and washed 3 times with deionized water each time for 5.5 min.
(9) The treated tissue is soaked in a 90% ethanol solution 7 min, and then the solution is changed into deionized water 5min, so that the treatment is carried out repeatedly for 5 times. Then soaked in deionized water for 30 min a.
(10) Performing freeze-drying treatment, pre-freezing at-70 ℃ for 4h, then putting into a freeze dryer for freeze-drying, and vacuumizing in the 2 nd stage, wherein the freeze-drying degree is as follows:
Example 3
The embodiment provides an ordered collagen scaffold material, and the preparation method thereof is as follows:
(1) Fresh fascia tissue was taken, physically shaved off muscle and adipose tissue, and soaked in deionized water at 4 ℃ for 2h, with water changed every half hour.
(2) After immersing the tissue in water at 4 ℃ for 15 minutes, the tissue is subjected to electric pulse stimulation by using an electrode, the stimulation voltage is set to 20V, the pulse length is 20ms, the pulse interval is 5s, and 10 pulse stimulations are performed.
(3) Immersing the treated tissue in water at 16 ℃ for ultrasonic stimulation with the power of 50W and the stimulation time of 7 min.
(4) The steps (2) and (3) are repeated 5 times.
(5) The treated tissue was soaked with 2.5M NaOH aqueous solution for 0.5 h, then the alkaline solution was removed and rinsed 10 times with deionized water 5 min each.
(6) The treated tissue is soaked in a mixed solution of ethanol and ethyl acetate (volume ratio is 1:1), soaked for 10h times at 16 ℃, and then washed with deionized water for 10 times, each time for 5: 5 min.
(7) The treated tissue is treated by supercritical carbon dioxide under the pressure of 40 MPa and the temperature of 40 ℃, the flow rate of the carbon dioxide is 4L/min, and the time is 20 min.
(8) The treated tissue was treated with 1mg/ml EDC and 0.5mg/ml NHS solution at 16deg.C for 1 h, and the tissue was collected and washed 3 times with deionized water each time for 5.5 min.
(9) The treated tissue is soaked in 75% ethanol solution 8 min, and then the solution is changed into deionized water 5min, so that the treatment is performed repeatedly for 7 times. Then soaked in deionized water for 30 min a.
(10) Performing freeze-drying treatment, pre-freezing at-70 ℃ for 4h, then putting into a freeze dryer for freeze-drying, and vacuumizing in the 2 nd stage, wherein the freeze-drying degree is as follows:
Example 4
This example provides an ordered collagen scaffold material, the preparation method of which differs from example 1 only in that in step (6) the isovolumetric substitution of ethyl acetate with acetone is performed, the other conditions being maintained.
Example 5
The present example provides an ordered collagen scaffold material, the preparation method of which differs from example 1 only in that in step (6) the ethanol is replaced by ethyl acetate in equal volume, the other conditions being kept unchanged.
Example 6
The present example provides an ordered collagen scaffold material, the preparation method of which differs from example 1 only in that the degree of lyophilization in step (10) is as follows:
Comparative example 1
The comparative example provides an ordered collagen scaffold material, the preparation method of which is different from that of example 1 only in that the soaking time in the step (6) is 10 h 30min, the treatment operation of the step (7) is absent, and other conditions are kept unchanged.
Comparative example 2
This comparative example provides an ordered collagen scaffold material, the preparation method of which differs from that of example 1 only in that the supercritical carbon dioxide treatment time in step (7) is 1h, and the treatment operation of step (6) is absent, and the other conditions remain unchanged.
Comparative example 3
This comparative example provides an ordered collagen scaffold material, the preparation method of which differs from example 1 only in that the operation of step (9) is absent, and the other conditions remain unchanged.
Comparative example 4
This comparative example provides an ordered collagen scaffold material prepared by the following method (treatment with tributyl phosphate aqueous solution instead of combined treatment of electrical and ultrasonic stimulation):
(1) Fresh fascia tissue was taken, physically shaved off muscle and adipose tissue, and soaked in deionized water at 4 ℃ for 2h, with water changed every half hour.
(2) Tissue was treated with 1% tributyl phosphate in water 48 h times, 10min each with deionized water.
(3) The treated tissue was soaked in 2M NaOH aqueous solution for 1h, then the alkali solution was removed and rinsed 10 times with deionized water 5min each.
(4) The treated tissue was soaked in a mixed solution of ethanol and ethyl acetate (volume ratio of 1:2) at 16 ℃ for 10h times, and then rinsed with deionized water for 5 times min each time.
(5) The treated tissue is treated by supercritical carbon dioxide under the pressure of 30 MPa and the temperature of 30 ℃, the flow rate of the carbon dioxide is 6L/min, and the time is 30 min.
(6) The treated tissue was treated with 1mg/ml EDC and 0.5mg/ml NHS solution for 1h, and the tissue was collected and washed 3 times with deionized water, 5min each.
(7) The treated tissue is soaked in 100% ethanol solution 5min, and then the solution is changed into deionized water 5min, so that the treatment is carried out repeatedly for 5 times. Then soaked in deionized water for 30min a.
(8) Freeze-drying treatment, pre-freezing at-70deg.C for 4h, freeze-drying in freeze dryer, and vacuumizing at stage 2 to obtain the final product.
Comparative example 5
This comparative example provides an ordered collagen scaffold material prepared by the following (trypsin treatment instead of combined treatment of electrical pulse stimulation and ultrasonic stimulation):
(1) Fresh fascia tissue was taken, physically shaved off muscle and adipose tissue, and soaked in deionized water at 4 ℃ for 2h, with water changed every half hour.
(2) The tissue was soaked in 1% trypsin for 2.5 h, 25 mmol/L Tris-HCl (pH 7.6) buffer, and then washed 5 times with deionized water, 10min each.
(3) The treated tissue was soaked in 2M NaOH aqueous solution for 1h, then the alkali solution was removed and rinsed 10 times with deionized water 5min each.
(4) The treated tissue was soaked in a mixed solution of ethanol and ethyl acetate (volume ratio of 1:2) at 16 ℃ for 10h times, and then rinsed with deionized water for 5 times min each time.
(5) The treated tissue is treated by supercritical carbon dioxide under the pressure of 30 MPa and the temperature of 30 ℃, the flow rate of the carbon dioxide is 6L/min, and the time is 30 min.
(6) The treated tissue was treated with 1mg/ml EDC and 0.5mg/ml NHS solution for 1h, and the tissue was collected and washed 3 times with deionized water, 5min each.
(7) The treated tissue is soaked in 100% ethanol solution 5min, and then the solution is changed into deionized water 5min, so that the treatment is carried out repeatedly for 5 times. Then soaked in deionized water for 30min a.
(8) Freeze-drying treatment, pre-freezing at-70deg.C for 4h, freeze-drying in freeze dryer, and vacuumizing at stage 2 to obtain the final product.
Comparative example 6
This comparative example provides an ordered collagen scaffold material prepared by the following method (combined treatment of papain and DNase I/RNase I treatment instead of electrical pulse stimulation and ultrasonic stimulation):
(1) Fresh fascia tissue was taken, physically shaved off muscle and adipose tissue, and soaked in deionized water at 4 ℃ for 2h, with water changed every half hour.
(2) The tissue was soaked in 300U/g papain 0.5 h in phosphate buffer ph=6 and then washed 5 times with deionized water, 10 min each. 3h was treated in 100U/g DNase I/RNase I mixture and then rinsed 5 times with deionized water, 10 min each.
(3) The treated tissue was soaked in 2M NaOH aqueous solution for 1h, then the alkali solution was removed and rinsed 10 times with deionized water 5min each.
(4) The treated tissue was soaked in a mixed solution of ethanol and ethyl acetate (volume ratio of 1:2) at 16 ℃ for 10h times, and then rinsed with deionized water for 5 times min each time.
(5) The treated tissue is treated by supercritical carbon dioxide under the pressure of 30 MPa and the temperature of 30 ℃, the flow rate of the carbon dioxide is 6L/min, and the time is 30 min.
(6) The treated tissue was treated with 1mg/ml EDC and 0.5mg/ml NHS solution for 1h, and the tissue was collected and washed 3 times with deionized water, 5min each.
(7) The treated tissue is soaked in 100% ethanol solution 5min, and then the solution is changed into deionized water 5min, so that the treatment is carried out repeatedly for 5 times. Then soaked in deionized water for 30min a.
(8) Freeze-drying treatment, pre-freezing at-70deg.C for 4h, freeze-drying in freeze dryer, and vacuumizing at stage 2 to obtain the final product.
Comparative example 7
This comparative example provides an ordered collagen scaffold material prepared by the following method (combined treatment of electrical pulse stimulation and ultrasonic stimulation replaced with sodium dodecyl sulfate and sodium deoxycholate treatment):
(1) Fresh fascia tissue was taken, physically shaved off muscle and adipose tissue, and soaked in deionized water at 4 ℃ for 2h, with water changed every half hour.
(2) The tissue was treated with 0.2% sodium dodecyl sulfate in water 0.5h, 2h with 3% sodium deoxycholate, and then rinsed 10 times with deionized water 5min each.
(3) The treated tissue was soaked in 2M NaOH aqueous solution for 1h, then the alkali solution was removed and rinsed 10 times with deionized water 5min each.
(4) The treated tissue was soaked in a mixed solution of ethanol and ethyl acetate (volume ratio of 1:2) at 16 ℃ for 10h times, and then rinsed with deionized water for 5 times min each time.
(5) The treated tissue is treated by supercritical carbon dioxide under the pressure of 30 MPa and the temperature of 30 ℃, the flow rate of the carbon dioxide is 6L/min, and the time is 30 min.
(6) The treated tissue was treated with 1mg/ml EDC and 0.5mg/ml NHS solution for 1h, and the tissue was collected and washed 3 times with deionized water, 5min each.
(7) The treated tissue is soaked in 100% ethanol solution 5min, and then the solution is changed into deionized water 5min, so that the treatment is carried out repeatedly for 5 times. Then soaked in deionized water for 30min a.
(8) Freeze-drying treatment, pre-freezing at-70deg.C for 4h, freeze-drying in freeze dryer, and vacuumizing at stage 2 to obtain the final product.
Comparative example 8
This comparative example provides an ordered collagen scaffold material prepared as follows (with a single ultrasonic stimulus):
(1) Fresh fascia tissue was taken, physically shaved off muscle and adipose tissue, and soaked in deionized water at 4 ℃ for 2h, with water changed every half hour.
(2) After soaking the tissue in water at 4 ℃ for 15 minutes, soaking the tissue in water at 16 ℃ for ultrasonic stimulation with power of 200W and stimulation time of 10min.
(3) The above step (2) is repeated 3 times.
(4) The treated tissue was soaked in 2M NaOH aqueous solution for 1h, then the alkali solution was removed and rinsed 10 times with deionized water 5min each.
(5) The treated tissue was soaked in a mixed solution of ethanol and ethyl acetate (volume ratio of 1:2) at 16 ℃ for 10h times, and then rinsed with deionized water for 5 times min each time.
(6) The treated tissue is treated by supercritical carbon dioxide under the pressure of 30 MPa and the temperature of 30 ℃, the flow rate of the carbon dioxide is 6L/min, and the time is 30 min.
(7) The treated tissue was treated with 1mg/ml EDC and 0.5mg/ml NHS solution for 1h, and the tissue was collected and washed 3 times with deionized water, 5min each.
(8) The treated tissue is soaked in 100% ethanol solution 5min, and then the solution is changed into deionized water 5min, so that the treatment is carried out repeatedly for 5 times. Then soaked in deionized water for 30min a.
(9) Freeze-drying treatment, pre-freezing at-70deg.C for 4h, freeze-drying in freeze dryer, and vacuumizing at stage 2 to obtain the final product.
Comparative example 9
This comparative example provides an ordered collagen scaffold material prepared as follows (using single electrical pulse stimulation):
(1) Fresh fascia tissue was taken, physically shaved off muscle and adipose tissue, and soaked in deionized water at 4 ℃ for 2h, with water changed every half hour.
(2) After the tissue is soaked in water at 4 ℃ for 15 minutes, the electrode is used for carrying out electric pulse stimulation on the tissue, the stimulation voltage is set to 180V, the pulse length is 200ms, the pulse interval is 1s, and 100 pulse stimulation is carried out.
(3) The above step (2) is repeated 3 times.
(4) The treated tissue was soaked in 2M NaOH aqueous solution for 1h, then the alkali solution was removed and rinsed 10 times with deionized water 5min each.
(5) The treated tissue was soaked in a mixed solution of ethanol and ethyl acetate (volume ratio of 1:2) at 16 ℃ for 10h times, and then rinsed with deionized water for 5 times min each time.
(6) The treated tissue is treated by supercritical carbon dioxide under the pressure of 30 MPa and the temperature of 30 ℃, the flow rate of the carbon dioxide is 6L/min, and the time is 30 min.
(7) The treated tissue was treated with 1mg/ml EDC and 0.5mg/ml NHS solution for 1h, and the tissue was collected and washed 3 times with deionized water, 5min each.
(8) The treated tissue is soaked in 100% ethanol solution 5min, and then the solution is changed into deionized water 5min, so that the treatment is carried out repeatedly for 5 times. Then soaked in deionized water for 30min a.
(9) Freeze-drying treatment, pre-freezing at-70deg.C for 4h, freeze-drying in freeze dryer, and vacuumizing at stage 2 to obtain the final product.
Test example 1
As shown in fig. 1, the collagen scaffold material according to the present invention has an orderly parallel fiber structure.
Test example 2
Whether neurons can grow on the collagen scaffold material related to the invention is examined, and the product prepared in the example 1is taken as an example, and the specific operation is as follows: embryonic rat spinal cord nerve stem cells pregnant for 14 days are isolated, balled in DMEM/F12 medium containing 20ng/mL bFGF and 20ng/mL EGF for 7 days, 1000 cells are inoculated on a 1 cm-long scaffold material, 10ng/mL NT3 and 20ng/mL BDNF of the cell culture medium are added for 7 days, cells are subjected to immunofluorescent staining by Tuj1 antibodies, and the adhesion and growth of Tuj1 positive neuron cells on the material are analyzed. The results are shown in FIG. 2 (scale of 100 μm), and it is understood that rat neuronal cells can grow on the collagen scaffold material according to the present invention in order along the extending direction of the material.
Test example 3
The following properties were tested on the products prepared in examples 1 to 6 and comparative examples 1 to 9, respectively, as shown in the following specific test methods or criteria, and the results are shown in Table 1:
(1) Nucleic acid content: the test was carried out according to the method specified in YY/T0606.25-2014.
(2) Fat content: according to the method specified in the first method of GB/T5009.6-2010.
(3) Standard collagen content: the test was performed according to the method for determining hydroxyproline B in appendix B of YY/T1511-2017.
(4) Cytotoxicity: the detection is carried out according to the method specified in GB/T16886.5-2017.
(5) Scar ratio detection after spinal cord injury zone transplantation:
(5.1) construction of adult rats (model SD rats, 10 weeks old) model of 5mm full-transection spinal cord injury at chest: rats were anesthetized with sodium pentobarbital, the dorsal spinal column of the spinal cord at thoracic segment T8 was opened, 5mm defects were made in spinal cord tissue, the tissue was hemostatic, and grafts of different materials were performed.
(5.2) Grafting the prepared ordered collagen scaffold material of each group to the spinal cord injury site, and taking injured rats without the transplanted material as a control, wherein 9 rats are used in each group. After 72 days of implantation, animals were euthanized, spinal cord tissue was taken and immersed in 4% formaldehyde solution for fixation for 48 hours. Then 30% sucrose solution was added for dehydration for 72 hours. The tissue was then embedded and frozen for sectioning to a section thickness of 20 μm. The sections were then immunofluorescent stained with antibody Anti-Chondroitin Sulfate antibody (CS-56), manufacturer abcam, cat No. ab11570. Meanwhile, the cell nuclei are stained by using DAPI, and the proportion of CS-56 positive areas in the spinal cord area is counted and used as the proportion of scars after the transplantation of the damaged area. The results of the material-free and example 1 material-free graft groups are shown in FIG. 3 (CS-56 is chondroitin sulfate, indicating formation of a glial scar, DAPI indicates nuclear staining, and dashed lines indicate lesion edge locations).
From the data in fig. 3 and table 1, it can be seen that the deposition of glial scar is significantly reduced after the ordered collagen scaffold material according to the present invention is transplanted to the spinal cord injury site, which is important for repairing spinal cord injury.
(7) Nerve regeneration length detection after spinal cord injury zone transplantation:
The sections were immunofluorescent stained with antibody anti-beta III Tubulin antibody (Tuj 1), manufacturer abcam under the designation ab18207. Meanwhile, the nuclei are stained by using DAPI, and the length of Tuj1 positive nerve axons in the spinal cord area in the damaged area is counted and used as the nerve regeneration length after the damaged area is transplanted. The results of the material-free and example 1 material-transplanted groups are shown in FIG. 4 (Tuj-1 represents nerve axons, DAPI represents nuclei, scale 1 mm).
From the data shown in fig. 4 and table 1, it can be seen that the ordered collagen scaffold material according to the present invention can effectively promote regeneration of nerve axons at the injured site after being transplanted to the injured site of spinal cord.
TABLE 1
(8) Animal locomotor activity score (BBB score):
For each group of rats, after transplanting the ordered collagen scaffold material, a motor behavior score (BBB score, for evaluation criteria see table 2) was performed, with higher scores representing better motor function recovery, and the results are shown in table 3:
TABLE 2
TABLE 3 Table 3
As can be seen from the data in table 3: compared with comparative examples 1-6, the ordered collagen scaffold material prepared by the preparation method provided by the invention can obviously improve the recovery of exercise function after being transplanted to the spinal cord injury part.
Application example 1
The application example provides an ordered collagen scaffold material loaded with a drug cetuximab, and the preparation method comprises the following steps:
The ordered nerve regeneration collagen scaffold material (length 5mm, diameter 2 mm) prepared in example 1 was added with 10. Mu.L of cetuximab liquid (commercial specification 100 mg/20 ml/bottle from mercon pharmaceutical company, germany) at a concentration of 2. Mu.g/mu.L, and the whole was absorbed by the nerve regeneration collagen scaffold, and incubated at 4℃for 60 min to prepare the drug-loaded ordered collagen scaffold material.
Test example 4
The following performance tests are carried out on the products prepared by the application examples, the specific test methods or standards are shown as follows, and the results are shown in Table 4:
(1) Scar ratio detection after spinal cord injury zone transplantation: the method of the application example was the same as that of test example 3, except that the ordered collagen scaffold material loaded with the drug cetuximab was transplanted to the spinal cord injury site. The results after material implantation repair are shown in FIG. 5 (CS-56 is chondroitin sulfate, indicating formation of glial scar, DAPI indicates nuclear staining, and dotted line indicates lesion edge location).
(2) Nerve regeneration length detection after spinal cord injury zone transplantation: the method of the application example was the same as that of test example 3, except that the ordered collagen scaffold material loaded with the drug cetuximab was transplanted to the spinal cord injury site. Application example the results after material implantation repair are shown in FIG. 6 (Tuj-1 represents the nerve axon, DAPI represents the nucleus, scale 1 mm).
From the data in table 4, it is clear that the ordered collagen scaffold material according to the present invention can further promote regeneration of nerve axons at the injured site after being transplanted to the spinal cord injured site.
TABLE 4 Table 4
The applicant states that the technical solution of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above embodiments, i.e. it does not mean that the present invention must be implemented by the above embodiments. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.