CN118873745B - A biological scaffold for repairing ligament damage and simultaneously loading PRP and BMSCs and a forming method thereof - Google Patents
A biological scaffold for repairing ligament damage and simultaneously loading PRP and BMSCs and a forming method thereof Download PDFInfo
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- CN118873745B CN118873745B CN202411364343.6A CN202411364343A CN118873745B CN 118873745 B CN118873745 B CN 118873745B CN 202411364343 A CN202411364343 A CN 202411364343A CN 118873745 B CN118873745 B CN 118873745B
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Abstract
本发明属于再生医学技术领域，具体涉及一种用于修复韧带损伤的同时负载PRP和BMSCs的生物支架及其成型方法。本发明所述生物支架以非溶解性I型胶原为主要原料制得，赋予其优异的力学性能和降解性能；该生物支架为圆柱形结构或腰型结构，在生物支架中沿生物支架的轴向均匀设置多个孔道结构，所述孔道结构贯穿生物支架，该生物支架采用与前交叉韧带结构更接近的纵向有序孔道，一方面模拟了前交叉韧带组织原有的生理结构，另一方面，可保证PRP和BMSCs的负载，该生物支架可促进胶原的分泌和细胞外基质的沉积，有效提高生物支架的完整性和力学强度，使该生物支架在修复韧带损伤领域具有良好的应用前景。
The present invention belongs to the field of regenerative medicine technology, and specifically relates to a bioscaffold for repairing ligament injuries and simultaneously loading PRP and BMSCs and a molding method thereof. The bioscaffold of the present invention is made of insoluble type I collagen as the main raw material, which gives it excellent mechanical properties and degradation properties; the bioscaffold is a cylindrical structure or a waist-shaped structure, and a plurality of pore structures are evenly arranged along the axial direction of the bioscaffold in the bioscaffold, and the pore structure runs through the bioscaffold. The bioscaffold adopts a longitudinal ordered pore that is closer to the anterior cruciate ligament structure, which simulates the original physiological structure of the anterior cruciate ligament tissue on the one hand, and on the other hand, it can ensure the loading of PRP and BMSCs. The bioscaffold can promote the secretion of collagen and the deposition of extracellular matrix, effectively improve the integrity and mechanical strength of the bioscaffold, and make the bioscaffold have good application prospects in the field of repairing ligament injuries.
Description
Technical Field
The invention belongs to the technical field of regenerative medicine, and particularly relates to a biological scaffold for repairing ligament injury and loading PRP and BMSCs and a forming method thereof.
Background
The cruciate ligament injury is mainly repaired by adopting a reconstruction operation under an arthroscope, the reconstruction ligament is mainly transplanted by an autologous patella tendon or popliteal tendon or an artificial ligament woven by a high polymer material, and the cruciate ligament is mainly transplanted and fixed in a cortical suspension fixation (a belt loop titanium plate) mode and an interface screw fixation mode. However, the anterior cruciate ligament has poor self-healing ability due to its own histological features, and if an artificial ligament is used, the anterior cruciate ligament has high price and good healing effect.
Both conventional anterior cruciate ligament suturing and reconstruction are simple suturing or implantation of artificial ligaments only at the site of injury, lacking a critical portion of the platelet clot as compared to the "fibrin-platelet clot" of human tissue. However, the platelet clot contains a large amount of biological factors such as TGF-beta, PDGF, bFGF, VEGF, EGF, IGF-1 and the like, and can promote the differentiation of bone marrow mesenchymal stem cells into fibrous cells, the synthesis of extracellular matrix and the like in a series of biochemical processes, so that the repair efficiency is improved. Thus, one key technique to achieve anterior cruciate ligament repair is whether a provisional ligament like a "fibrin-platelet clot" can be constructed at the lesion to promote self-repair of the anterior cruciate ligament.
At present, although the regeneration technology of repairing ligaments by using the biological scaffold load-related repair factors exists, the research in the field still has the technical problems of unstable mechanical properties of biological scaffold raw materials, single load substances, poor load absorption capacity, platelet coagulation, complex forming process and the like.
Disclosure of Invention
Based on the above technical background, the main objective of the present invention is to provide a biological scaffold for repairing ligament injury and loading PRP (platelet rich plasma) and BMSCs (bone marrow mesenchymal stem cells) at the same time and a molding method thereof, so as to overcome the defects in the prior art.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention comprises the following steps:
The first aspect of the invention is to provide a biological scaffold for repairing ligament injury and simultaneously loading PRP and BMSCs, wherein the biological scaffold for repairing ligament injury and simultaneously loading PRP and BMSCs is prepared by taking insoluble type I collagen as a main raw material;
The biological stent is of a cylindrical structure or a waist-shaped structure, a plurality of pore canal structures are uniformly arranged in the biological stent along the axial direction of the biological stent, and the pore canal structures penetrate through the biological stent.
Preferably, the cross section of the pore canal structure is circular or polygonal.
Preferably, the pore structures are each less than 300 μm in diameter.
A second aspect of the present invention is to provide a method for forming a biological scaffold for repairing ligament injury while loading PRP and BMSCs according to the first aspect of the present invention, the method comprising the steps of:
In the step (1) of the process,
Preferably, the acidic solution is selected from one or more of acetic acid, lactic acid and hydrochloric acid, and/or,
Preferably, the pH value of the acidic solution is 2-3.5, and/or,
Preferably, the suspension has a solids content of 0.3 to 2%, and/or,
Preferably, the suspension is sheared and dispersed for 120-240 min at a shearing speed of 10000-25000 rpm, and after shearing and dispersing, an alkaline solution with a pH of 9-11 is adopted to adjust the pH to 4.5-5.5 under the condition of 0-4 ℃.
Preferably, the alkali solution is selected from one or more of ammonia water, sodium bicarbonate solution and sodium hydroxide solution, and/or,
Preferably, the suspension after pH adjustment is centrifuged for 10-30 min at 0-8 ℃ and 8000-50000 rpm to concentrate the suspension, the concentration after concentration is 5-15%, and/or,
Preferably, the pre-freezing condition is that the concentrated product is placed on the surface of a refrigerant, and is vertically frozen at a speed of 1-7 mm/min, wherein the vertical freezing time is 5-75 min.
In the step 2 of the process, the process is carried out,
Preferably, the freeze drying comprises four stages, each stage in the freeze drying process is performed under the vacuum condition of 0.1-0.5 mbar, and the freeze drying procedure is as follows:
The first stage, cooling the pre-frozen biological scaffold from room temperature to 0-4 ℃, wherein the cooling time is 20-60 min, and preserving heat for 90-180 min at 0-4 ℃;
the second stage, namely cooling from 0-4 ℃ to-30 to-50 ℃ for 60-120 min, and preserving heat for 40-120 min at-30 to-50 ℃;
The third stage, heating from-30 to-50 ℃ to 0-4 ℃ for 30-90 min, and preserving heat for 960-1680 min at 0-4 ℃;
and in the fourth stage, the temperature is increased from 0-4 ℃ to 15-22 ℃, the heating time is 20-60 min, the temperature is kept at 15-22 ℃ for 20-90 min, and the non-crosslinked biological scaffold is obtained after freeze drying.
More preferably, the non-crosslinked biological scaffold is placed under the condition of negative one atmosphere pressure and 100-120 ℃ for heat preservation for 24-75 hours, and the physical crosslinked biological scaffold is obtained.
More preferably, EDC and NHS are prepared according to the mol ratio (2-8): 1 to obtain a reaction solution, the non-crosslinked biological scaffold is soaked in the reaction solution, and the reaction is carried out for 4-12 hours at the temperature of 2-5 ℃ to obtain the chemically crosslinked biological scaffold.
The invention has the beneficial effects that:
(1) The biological scaffold adopts the insoluble type I collagen which can keep a triple helix structure as a main raw material, and has more excellent mechanical property and higher degradation rate compared with other biological scaffolds prepared by using soluble collagen.
(2) The existing biological scaffold mostly adopts a disordered porous structure similar to a sponge structure, and the biological scaffold adopts a longitudinal ordered pore canal which is closer to an anterior cruciate ligament structure, so that on one hand, the original physiological structure of the anterior cruciate ligament tissue is simulated, and on the other hand, the simultaneous loading of PRP and BMSCs can be ensured. The ordered porous structure can induce the differentiation of fibroblast of BMSCs, promote the migration and arrangement of cells, promote the secretion of collagen and the deposition of extracellular matrix, and effectively improve the integrity and mechanical strength of the biological scaffold.
(3) The biological scaffold has excellent liquid absorptivity, PRP and BMSCs can be loaded simultaneously in the using process, the original fibrin-platelet clot in the tissue is simulated, and the regeneration of the damaged anterior cruciate ligament is promoted.
(4) The molding process of the biological scaffold can adjust the structure and the composition of the obtained product only by adjusting the centrifugation time and the concentration times of centrifugation concentration, so that the biological scaffold with different porosities and degradation performances is obtained, can be applied to anterior cruciate ligament injuries with different degrees, simplifies the traditional complex molding process, has mild production conditions, reduces the production difficulty, and all performance indexes of the obtained product meet the use requirements.
Drawings
FIG. 1 shows a schematic structural view of a biological stent according to the present invention;
FIG. 2 shows a process flow diagram of the biological stent of the present invention;
fig. 3 shows stress-strain curves of the bioscaffold according to the invention.
Detailed Description
The features and advantages of the present invention will become more apparent and evident from the following detailed description of the invention.
In a first aspect, the present invention provides a biological scaffold for repairing ligament injury while loading PRP and BMSCs, which is prepared from insoluble type I collagen as a main raw material, wherein the insoluble type I collagen can maintain a triple helix structure.
The biological support is of a cylindrical structure or a waist-shaped structure, the diameter of the two sections of the waist-shaped structure is larger than that of the middle section, a plurality of pore canal structures are uniformly arranged in the biological support along the axial direction of the biological support, the pore canal structures are parallel to each other, and the pore canal structures penetrate through the biological support.
Preferably, the cross section of the pore canal structure is circular or polygonal.
Preferably, the pore structures are each less than 300 μm in diameter.
The biological bracket is arranged into a waist-shaped structure, the diameters of the sections at the two ends of the biological bracket are larger, the contact area with a bone surface can be increased, the biological bracket is more firmly fixed, and meanwhile, the regeneration factor can be loaded in a larger area, so that the regeneration effect is better.
The collagen content of the biological scaffold is more than or equal to 98%, the liquid absorptivity is more than or equal to 1000%, and the pore diameter is less than or equal to 300 mu m.
A second aspect of the present invention is to provide a method for forming a biological scaffold for repairing ligament injury while loading PRP and BMSCs according to the first aspect of the present invention, the method comprising the steps of:
step 1, dispersing insoluble type I collagen in an acidic solution to obtain a suspension, shearing and dispersing the suspension, adjusting pH, and then centrifuging, concentrating and pre-freezing to obtain a pre-frozen biological scaffold;
and 2, freeze-drying the pre-frozen biological scaffold to obtain the biological scaffold.
The above steps are specifically described below.
In step 1, the acidic solution is one or more selected from acetic acid, lactic acid and hydrochloric acid, preferably, the acidic solution is acetic acid, lactic acid or hydrochloric acid.
The pH value of the acidic solution is 2-3.5, preferably 2.5-3.2.
The solid content of the suspension is 0.3-2%, preferably the solid content of the suspension is 0.5-1.5%.
And shearing and dispersing the suspension at a shearing speed of 10000-25000 rpm for 120-240 min, and adjusting the pH to 4.5-5.5 by adopting an alkali solution with the pH of 9-11 under the condition of 0-4 ℃ after shearing and dispersing.
Preferably, the suspension is sheared and dispersed for 180min at a shearing speed of 12000-20000 rpm, and after shearing and dispersing, the pH is adjusted to 4.6-4.8 by adopting an alkali solution with the pH of 10 under the condition of 0 ℃.
The alkali solution is selected from one or more of ammonia water, sodium bicarbonate solution and sodium hydroxide solution.
Preferably, the alkaline solution is aqueous ammonia, sodium bicarbonate solution or sodium hydroxide solution.
And centrifuging the suspension after pH adjustment at 0-8 ℃ and 8000-50000 rpm for 10-30 min to concentrate, wherein the concentration after concentration is 5-15%.
Preferably, the suspension after pH adjustment is centrifuged for 10-20 min at 4-6 ℃ and 10000-20000 rpm, and the concentration after concentration is 10%.
The pre-freezing condition is that the concentrated product is placed on the surface of a refrigerant, vertical freezing is carried out at a speed of 1-7 mm/min, and the vertical freezing time is 5-75 min.
Preferably, the prefreezing condition is that the concentrated product is placed on the surface of liquid nitrogen, and is subjected to vertical freezing at a speed of 1-5 mm/min, wherein the vertical freezing time is 10-60 min.
The pre-freezing stage is critical to the formation of vertical tunnels, the concentration step is performed by using a separable polytetrafluoroethylene or metal centrifuge tube, the centrifuge tube is lowered to the surface of a refrigerant (liquid nitrogen or other refrigerants are selected, preferably liquid nitrogen, because liquid nitrogen can provide more supercooling degree), then the liquid nitrogen is lowered at a speed of 1-5 mm/min for vertical freezing, 10-60 min is remained, the centrifuge tube is opened after the pre-freezing is finished, and a sample is taken out for a subsequent freeze drying process.
In the step 2, the freeze drying comprises four stages, wherein each stage in the freeze drying process is performed under the vacuum condition of 0.1-0.5 bar, and the freeze drying process comprises the following steps:
The first stage, cooling the pre-frozen biological scaffold from room temperature to 0-4 ℃, wherein the cooling time is 20-60 min, and preserving heat for 90-180 min at 0-4 ℃;
the second stage, namely cooling from 0-4 ℃ to-30 to-50 ℃ for 60-120 min, and preserving heat for 40-120 min at-30 to-50 ℃;
The third stage, heating from-30 to-50 ℃ to 0-4 ℃ for 30-90 min, and preserving heat for 960-1680 min at 0-4 ℃;
and a fourth stage, namely heating from 0-4 ℃ to 15-22 ℃, wherein the heating time is 20-60 min, and preserving heat for 20-90 min at 15-22 ℃. And freeze-drying to obtain the non-crosslinked biological scaffold.
Preferably, the freeze-drying comprises four stages, each stage of the freeze-drying process being carried out under vacuum conditions of 0.3mbar, the procedure of freeze-drying being:
the first stage, cooling the pre-frozen biological scaffold from room temperature to 0 ℃, wherein the cooling time is 30-60 min, and preserving heat for 120-180 min at 0 ℃;
the second stage, namely cooling from 0 ℃ to-40 ℃ for 80-120 min, and preserving heat for 60-120 min at-40 ℃;
the third stage, heating from-40 ℃ to 0 ℃, wherein the heating time is 40-90 min, and preserving heat at 0 ℃ for 1080-1680 min;
And a fourth stage, namely heating from 0 ℃ to 20 ℃, wherein the heating time is 30-60 min, and preserving heat for 30-60 min at 20 ℃. And freeze-drying to obtain the non-crosslinked biological scaffold.
According to the preferred embodiment of the invention, the non-crosslinked biological scaffold is placed under the condition of negative one atmosphere pressure and 100-120 ℃ for heat preservation for 24-75 hours, and the physical crosslinked biological scaffold is obtained.
More preferably, the non-crosslinked bioscaffold is incubated at a temperature of 105 ℃ for 72 hours at a negative one atmosphere to provide a physically crosslinked bioscaffold.
According to the preferred embodiment of the invention, EDC (1-ethyl- (3-dimethylaminopropyl) carbodiimide) and NHS (N-hydroxy thiosuccinimide) are prepared according to the mol ratio (2-8): 1 to obtain a reaction solution, and the non-crosslinked biological scaffold is soaked in the reaction solution and reacts for 4-12 hours at the temperature of 2-5 ℃ to obtain the chemically crosslinked biological scaffold.
More preferably, EDC and NHS are prepared in a molar ratio of 4:1 to give a reaction solution, and the non-crosslinked bioscaffold is immersed in the reaction solution and reacted at 4 ℃ to 8 h. Thus obtaining the chemically crosslinked biological scaffold.
Examples
The invention is further illustrated by the following specific examples, which are intended to be illustrative of the invention and are not intended to limit the scope of the invention. The raw materials adopted in the embodiment of the invention are all purchased.
Example 1
The biological scaffold for repairing ligament injury and loading PRP and BMSCs is prepared from insoluble type I collagen serving as a main raw material, the biological scaffold is of a cylindrical structure, the overall size is 44 mm (height) multiplied by 22 mm (diameter), the biological scaffold is uniformly arranged in the biological scaffold along the axial direction of the biological scaffold, the pore channel structure penetrates through the biological scaffold, the section of the pore channel structure is circular, and the diameters of the pore channel structures are all less than or equal to 300 mu m, as shown in figure 1.
Example 2
The 5g non-soluble type I collagen was dispersed in 1000 mL, ph=3.2 glacial acetic acid solution at a solids content of 0.5% and the suspension was subsequently shear dispersed 180 min using a high speed shear at a speed of 12000 rpm at 0 ℃. After the completion of the dispersion, the pH of the dispersion was adjusted to 4.7 using dilute aqueous ammonia at ph=10 at 0 ℃. The suspension was concentrated by centrifugation at 12000 rpm at 10 min using a low temperature high speed centrifuge at 6 ℃. The centrifuge tube used for concentration was placed on the surface of liquid nitrogen, then subjected to vertical freezing (prefreezing) at a rate of 2 mm/min, left for 15: 15 min, opened after prefreezing, and the sample was taken out for freeze-drying by the following procedure:
(1) Cooling to 0 ℃ at room temperature and keeping the temperature constant, wherein the cooling process is 30 min, and the constant temperature process is 120 min;
(2) Cooling to-40 ℃ at 0 ℃ and keeping the temperature constant, wherein the cooling process is 80 min, and the constant temperature process is 60 min;
(3) Heating to-40 ℃ to 0 ℃ and keeping the temperature constant, wherein the heating process is 40 min, and the constant temperature process is 1080min;
(4) The temperature is raised to 20 ℃ at 0 ℃, the heating time is 30 min, the constant temperature time is 30 min, and the vacuum degree of each step is kept at 0.3 mbar. And obtaining the non-crosslinked biological scaffold after the freeze-drying procedure is completed.
Example 3
The insoluble type I collagen of 7.5 g was dispersed in a lactic acid solution of 1000 mL, ph=3.2, at which point the solids content of the suspension was 0.75%, and then the suspension was shear dispersed 180 min using a high speed shear at a speed of 12000 rpm under conditions of 0 ℃. After the completion of the dispersion, the pH of the dispersion was adjusted to 4.8 using a sodium bicarbonate solution with ph=10 at 0 ℃. The above suspension was concentrated by centrifugation at 15 min using a low temperature high speed centrifuge at a speed of 10000 rpm at 4 ℃. The centrifuge tube used for concentration was placed on the surface of liquid nitrogen, then frozen vertically (pre-frozen) at a rate of 3 mm/min, left for 10: 10 min, opened after the pre-freezing, and the sample was taken out for freeze-drying by the following procedure:
(1) Cooling to 0 ℃ at room temperature and keeping the temperature constant, wherein the cooling process is 40 min, and the constant temperature process is 150 min;
(2) Cooling to-40 ℃ at 0 ℃ and keeping the temperature constant, wherein the cooling process is 80 min, and the constant temperature process is 80 min;
(3) -40 ℃ to 0 ℃ and keeping constant temperature, wherein the temperature rising process is 60 min, and the constant temperature process is 1320 min;
(4) The temperature is raised to 20 ℃ at 0 ℃, the heating time is 40 min, the constant temperature time is 40 min, and the vacuum degree of each step is kept at 0.3 mbar. And obtaining the non-crosslinked biological scaffold after the freeze-drying procedure is completed.
Example 4
15G of insoluble type I collagen was dispersed in 1000 mL, pH=2.5 hydrochloric acid solution at a solids content of 1.5%, and the suspension was subsequently shear-dispersed 180 min at a speed of 20000 rpm using a high speed shear at 0 ℃. After the completion of the dispersion, the pH of the dispersion was adjusted to 4.6 using a sodium hydroxide solution having ph=10 at 0 ℃. The suspension was concentrated by centrifugation at 20 min at 20000 rpm using a low temperature high speed centrifuge at 4 ℃. The centrifuge tube used for concentration was placed on the surface of liquid nitrogen, then frozen vertically (pre-frozen) at a rate of 1 mm/min, left for 10: 10 min, opened after the pre-freezing, and the sample was taken out for freeze-drying by the following procedure:
(1) Cooling to 0 ℃ at room temperature and keeping the temperature constant, wherein the cooling process is 60 min, and the constant temperature process is 180 min;
(2) Cooling to-40 ℃ at 0 ℃ and keeping the temperature constant, wherein the cooling process is 120 min, and the constant temperature process is 120 min;
(3) -40 ℃ to 0 ℃ and keeping constant temperature, wherein the temperature rising process is 90 min, and the constant temperature process is 1680 min;
(4) The temperature is raised to 20 ℃ at 0 ℃, the heating time is 60min, the constant temperature time is 60min, and the vacuum degree of each step is kept at 0.2 mbar. And obtaining the non-crosslinked biological scaffold after the freeze-drying procedure is completed.
Example 5
The 5g non-soluble type I collagen was dispersed in 1000 mL, ph=3.2 glacial acetic acid solution at a solids content of 0.5% and the suspension was subsequently shear dispersed 180 min using a high speed shear at a speed of 12000 rpm at 0 ℃. After the completion of the dispersion, the pH of the dispersion was adjusted to 4.7 using dilute aqueous ammonia at ph=10 at 0 ℃. The suspension was concentrated by centrifugation at 12000 rpm at 10 min using a low temperature high speed centrifuge at 6 ℃. The centrifuge tube used for concentration was placed on the surface of liquid nitrogen, then subjected to vertical freezing (prefreezing) at a speed of 5 mm/min, left for 60: 60min, opened after prefreezing, and the sample was taken out for freeze-drying by the following procedure:
(1) Cooling to 0 ℃ at room temperature and keeping the temperature constant, wherein the cooling process is 30 min, and the constant temperature process is 120 min;
(2) Cooling to-40 ℃ at 0 ℃ and keeping the temperature constant, wherein the cooling process is 80 min, and the constant temperature process is 60 min;
(3) -40 ℃ to 0 ℃ and keeping constant temperature, wherein the temperature rising process is 40 min, and the constant temperature process is 1080 min;
(4) The temperature is raised to 20 ℃ at 0 ℃, the heating time is 30min, the constant temperature time is 30min, and the vacuum degree of each step is kept at 0.3 mbar.
And (3) placing the freeze-dried product into a vacuum oven, adjusting the vacuum degree to minus one atmosphere, the temperature to 105 ℃ and the time to 72 h, and obtaining the physical cross-linked biological scaffold after the reaction is completed.
Example 6
The 5g non-soluble type I collagen was dispersed in 1000 mL, ph=3.2 glacial acetic acid solution at a solids content of 0.5% and the suspension was subsequently shear dispersed 180 min using a high speed shear at a speed of 12000 rpm at 0 ℃. After the completion of the dispersion, the pH of the dispersion was adjusted to 4.7 using dilute aqueous ammonia at ph=10 at 0 ℃. The suspension was concentrated by centrifugation at 12000 rpm at 10 min using a low temperature high speed centrifuge at 6 ℃. The centrifuge tube used for concentration was placed on the surface of liquid nitrogen, then frozen vertically (pre-frozen) at a rate of 4 mm/min, left for 30min, opened after the pre-freezing is completed, and the sample was taken out for freeze-drying by the following procedure:
(1) Cooling to 0 ℃ at room temperature and keeping the temperature constant, wherein the cooling process is 30 min, and the constant temperature process is 120 min;
(2) Cooling to-40 ℃ at 0 ℃ and keeping the temperature constant, wherein the cooling process is 80 min, and the constant temperature process is 60 min;
(3) -40 ℃ to 0 ℃ and keeping constant temperature, wherein the temperature rising process is 40 min, and the constant temperature process is 1080 min;
(4) The temperature is raised to 20 ℃ at 0 ℃, the heating time is 30min, the constant temperature time is 30min, and the vacuum degree of each step is kept at 0.3 mbar.
The EDC and NHS were prepared in a molar ratio of 4:1, and the freeze-dried product was immersed therein and reacted at 4 ℃ for 8 h. The chemically crosslinked biological scaffold can be obtained.
Experimental example
Experimental example 1 mechanical Property test
The mechanical properties of the collagen scaffolds were tested by testing tensile strength. The specific test procedure is that the bracket sample prepared in the example 2 is cut into a long strip shape with the size of 50 multiplied by 10 mm, the thickness of the long strip shape is measured by an electronic universal tester, the two ends of the sample are fixed by a clamp of the electronic universal tester, and the tensile strength of the bracket material is detected at the tensile rate of 1 mm/min. Three experiments were repeated for 3 samples of each set of scaffold material, averaged. The calculation formula is ts=f/S.
Wherein, ts is tensile strength, F is the maximum tensile force applied by the material during fracture, N, S is the area of the fracture section of the material, and mm 2. The stress strain curve is shown in fig. 3.
Through testing, according to fig. 3 and a calculation formula, the tensile modulus of the biological scaffold prepared in example 2 is 15.200MPa, and the elongation at break is 51.7%. The biological scaffold has excellent mechanical properties.
Experimental example 2 degradation Rate test
The testing procedure is that a type I collagenase solution with the concentration of 30U/mL is prepared by using PBS buffer solution as degradation solution. About 50 mg of the sample of biological scaffold material prepared in example 2 was weighed and placed in a test tube. The material sample was completely immersed by adding 20 mL of the formulated degradation solution. The test tube was sealed and placed in a 37 ℃ thermostat water bath. 48 After h, the remaining undegraded biological scaffold material was lyophilized and weighed, the experiment was repeated three times, and the degradation rate was calculated according to the following formula:
D(%) =(W1 − W2)/W1× 100
wherein D is degradation rate, W1 is weight before degradation, and W2 is weight after degradation.
The test results are shown in table 1:
TABLE 1
As can be seen from Table 1, the degradation rate was 50% -70% when the degradation time was 12 h, 60% -75% when the degradation time was 24h, and 90% -100% when the degradation time was 36 h. The biological scaffold material can be completely degraded, and the degradation rate is higher.
The invention has been described in detail in connection with the specific embodiments and exemplary examples thereof, but such description is not to be construed as limiting the invention. It will be understood by those skilled in the art that various equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its embodiments without departing from the spirit and scope of the present invention, and these fall within the scope of the present invention. The scope of the invention is defined by the appended claims.
Claims (6)
1. The molding method of the biological scaffold for repairing ligament injury and simultaneously loading PRP and BMSCs is characterized in that the biological scaffold for repairing ligament injury and simultaneously loading PRP and BMSCs is prepared by taking insoluble type I collagen as a main raw material;
the biological bracket is of a cylindrical structure or a waist-shaped structure, a plurality of pore canal structures are uniformly arranged in the biological bracket along the axial direction of the biological bracket, and the pore canal structures penetrate through the biological bracket;
the molding method comprises the following steps:
step 1, dispersing insoluble type I collagen in an acidic solution to obtain a suspension, shearing and dispersing the suspension, adjusting pH, and then centrifuging, concentrating and pre-freezing to obtain a pre-frozen biological scaffold;
step 2, freeze-drying the pre-frozen biological scaffold to obtain the biological scaffold;
In the step 1, shearing and dispersing the suspension at a shearing speed of 10000-25000 rpm for 120-240 min, and adjusting the pH to 4.5-5.5 by adopting an alkali solution with the pH of 9-11 under the condition of 0-4 ℃ after shearing and dispersing;
the alkali solution is selected from one or more of ammonia water, sodium bicarbonate solution and sodium hydroxide solution;
Centrifuging the suspension after pH adjustment at 0-8 ℃ and 8000-50000 rpm for 10-30 min to concentrate, wherein the concentration after concentration is 5-15%;
The pre-freezing condition is that the concentrated product is placed on the surface of a refrigerant, and is vertically frozen at a speed of 1-7 mm/min, wherein the vertical freezing time is 5-75 min;
in the step 2, the freeze drying comprises four stages, wherein each stage in the freeze drying process is performed under the vacuum condition of 0.1-0.5 bar, and the freeze drying process comprises the following steps:
The first stage, cooling the pre-frozen biological scaffold from room temperature to 0-4 ℃, wherein the cooling time is 20-60 min, and preserving heat for 90-180 min at 0-4 ℃;
the second stage, namely cooling from 0-4 ℃ to-30 to-50 ℃ for 60-120 min, and preserving heat for 40-120 min at-30 to-50 ℃;
The third stage, heating from-30 to-50 ℃ to 0-4 ℃ for 30-90 min, and preserving heat for 960-1680 min at 0-4 ℃;
and in the fourth stage, the temperature is increased from 0-4 ℃ to 15-22 ℃, the heating time is 20-60 min, the temperature is kept at 15-22 ℃ for 20-90 min, and the non-crosslinked biological scaffold is obtained after freeze drying.
2. The molding method as claimed in claim 1, wherein,
The section of the pore canal structure is circular or polygonal.
3. The molding method as claimed in claim 1, wherein,
The diameter of the pore canal structure is less than 300 mu m.
4. The molding method as claimed in claim 1, wherein, in step 1,
The acidic solution is selected from one or more of acetic acid, lactic acid and hydrochloric acid, and/or,
The pH value of the acidic solution is 2-3.5, and/or,
The solid content of the suspension is 0.3-2%.
5. The molding method as claimed in claim 1, wherein, in step 2,
And placing the non-crosslinked biological scaffold at the condition of negative one atmosphere and at the temperature of 100-120 ℃ for heat preservation for 24-75 hours to obtain the physical crosslinked biological scaffold.
6. The molding method as claimed in claim 1, wherein, in step 2,
And preparing EDC and NHS according to the mol ratio of (2-8): 1 to obtain a reaction solution, soaking the non-crosslinked biological scaffold in the reaction solution, and reacting for 4-12 hours at the temperature of 2-5 ℃ to obtain the chemically crosslinked biological scaffold.
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