Method for preparing a composite biological scaffold for promoting anterior cruciate ligament repair and product thereof

Abstract

The application provides a preparation method of a composite biological scaffold for promoting anterior cruciate ligament repair and a product thereof, and the preparation method comprises the following steps: (1) selecting left-handed polylactic acid or polylactic acid-glycolic acid copolymer with a molecular weight of 250-500,000 to prepare a polylactic acid solution with a concentration of 2-5%, and then performing vacuum defoaming, sealing, slow pre-freezing and freeze-drying to obtain a polylactic acid sponge scaffold; (2) selecting type I or type III collagen with a molecular weight of 50-300,000 to prepare a collagen solution with a concentration of 1-3%, adjusting the pH value of the collagen solution to 6-8, preparing a ruthenium-peroxy disulfate solution, and mixing the collagen solution and the ruthenium-peroxy disulfate solution to obtain a mixed solution; (3) soaking the polylactic acid sponge scaffold in the mixed solution in step (2) under vacuum conditions; and (4) taking out the polylactic acid sponge scaffold, performing in-situ crosslinking through visible light irradiation, and obtaining a composite biological scaffold.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedicine, and particularly relates to a preparation method of a composite biological scaffold for promoting anterior cruciate ligament repair and a product thereof. BACKGROUND

[0002] There are four ligaments in the knee joint, including the medial collateral ligament, the lateral collateral ligament, the anterior cruciate ligament and the posterior cruciate ligament.

[0003] Anterior cruciate ligament injury is a common sports injury, which affects joint stability after injury, and further causes injury to other structures in the joint such as meniscus and articular cartilage, and eventually develops into irreversible osteoarthritis, which seriously affects daily life. There are two common surgical treatment methods in clinic: one is to use a substitute to reconstruct the anterior cruciate ligament; the other is to repair the anterior cruciate ligament by in-situ suture. Anterior cruciate ligament repair is a method of in-situ suture of the broken ligament stump. Compared with the anterior cruciate ligament reconstruction method, the anterior cruciate ligament repair method has smaller wound, is conducive to the preservation of the proprioception and function of the knee joint to a greater extent, and reduces the occurrence of arthritis, and thus is a more promising repair method. However, the anterior cruciate ligament is an intra-articular ligament surrounded by synovial fluid containing protease precursors (including plasminogen), and plasminogen is inactive in the uninjured joint. However, injury can cause the up-regulation of urokinase plasminogen activator (uPa) produced by synoviocytes, which cleaves plasminogen and forms plasmin. The ligament fracture surface will be attacked by synovial fluid and enzymes during the healing period, resulting in poor actual application effect of the in-situ suture repair method and a high surgical failure rate.

[0004] In recent years, it has become a research trend to use tissue engineering methods to construct biocompatible biological scaffolds to promote the repair and regeneration of damaged and defective tissues. For example: Xing Shuai, Xia Yayi, etc. disclosed a polylactic acid / silk fibroin composite tissue engineering scaffold for repairing cartilage defects, which solved the problem of poor biocompatibility of pure polylactic acid (PLA for short) scaffold by introducing silk fibroin. However, silk fibroin is basically only attached to the surface of the PLA scaffold, forming a silk fibroin layer on the surface of the PLA scaffold, and the porosity of the prepared composite biological scaffold is > 90%; Gu Zeming, etc. realized the formation of double tyrosine bonds in silk fibroin through visible light-mediated photo-oxidation-reduction system, realized the rapid crosslinking (<1min) of silk fibroin to form silk fibroin hydrogel, and also improved the mechanical properties of silk fibroin hydrogel; but the porosity of the silk fibroin hydrogel is not much affected, and the porosity of the prepared silk fibroin hydrogel is more than 90%; in addition, the Chinese patent with the authorization announcement number CN113301928B also discloses a biological scaffold, which includes a main body with a non-reconstituted collagen matrix, a biological material layer is coated on one side of the main body in a reduced pressure environment, and a tissue adhesive layer is coated on the other side of the main body. When the biological scaffold is adhered to the tissue through the tissue adhesive layer, a plurality of cells move from the tissue to the adhesive layer, the main body and the biological material layer in sequence to repair or regenerate the tissue. Although the porosity of the biological scaffold is not disclosed in the patent, from the mechanism of action, in order to realize the cell movement and nutrient transport and tissue metabolism, the biological scaffold must have a high porosity, and the higher the porosity, the better. The existing biological scaffolds (including the above two biological scaffolds) basically embed the defect site, guide the migration of surrounding cells into the internal pores of the biological scaffold to form tissue. The main role of the biological scaffold is: for cell inoculation, providing a support carrier and a nutrient source for cell growth; at the same time, providing a favorable channel for nutrient transport and tissue metabolism. Therefore, the existing biological scaffolds require higher porosity (usually more than 80%). However, the existing biological scaffolds are not ideal for the repair of the anterior cruciate ligament. SUMMARY

[0005] The purpose of the present application is to provide a preparation method of a composite biological scaffold for promoting the repair of the anterior cruciate ligament.

[0006] The preparation method of the composite biological scaffold for promoting the repair of the anterior cruciate ligament comprises the following steps:

[0007] (1) dissolve left-handed polylactic acid (PLLA) or polylactic acid-glycolic acid copolymer (PLGA) with a molecular weight of 250-500 thousand in an organic solvent to obtain a polylactic acid solution with a concentration of 2%-5%, pour the obtained polylactic acid solution into a mold, vacuum degassing, then seal, slow pre-freezing and freeze-drying to obtain a polylactic acid sponge scaffold;

[0008] (2) dissolve type I or type III collagen with a molecular weight of 50-300 thousand in dilute acetic acid to obtain a collagen solution with a concentration of 1%-3%; dissolve a transition metal complex ruthenium (Ru) and sodium peroxodisulfate in deionized water to obtain a ruthenium-sodium peroxodisulfate solution; add alkali to the collagen solution to adjust the pH value to obtain a collagen solution with a pH value of 6-8; mix the obtained ruthenium-sodium peroxodisulfate solution with the collagen solution with a pH value of 6-8, and stir uniformly to obtain a mixed solution;

[0009] (3) soak the polylactic acid sponge scaffold obtained in step (1) in the mixed solution obtained in step (2), the mixed solution completely submerges the polylactic acid sponge scaffold, and the soaking is carried out under vacuum for 3-12 hours, and the vacuum degree is controlled at 1x10 -6 ~ 1x10 -7 Pa;

[0010] (4) take out the polylactic acid sponge scaffold soaked in step (3), and irradiate it with visible light for in-situ crosslinking to obtain a composite biological scaffold.

[0011] The preparation method of the composite biological scaffold for promoting anterior cruciate ligament repair has the following advantages:

[0012] (1) The polylactic acid sponge scaffold is used as a "skeleton" in the present application, and polylactic acid with a molecular weight of 250-500 thousand is selected, and the concentration of the polylactic acid solution is controlled at 2%-5%, so as to avoid too low concentration which makes it difficult to form a sponge scaffold, and too high concentration which makes the scaffold pores too dense and is not conducive to collagen filling; then collagen is added to form a composite biological scaffold, polylactic acid and collagen are both high molecular materials with good biocompatibility, and are materials approved by the Food and Drug Administration (FDA) for implantation into the human body, and have no cytotoxicity and good biocompatibility; and the degradation period of polylactic acid meets the healing period of the anterior cruciate ligament, so that it can protect the ligament during repair for a long time;

[0013] (2) The polylactic acid sponge scaffold is soaked in the mixed solution containing collagen in the present application, so that collagen molecules are embedded in the internal pores of the polylactic acid sponge scaffold; in order to achieve good embedding and filling, the present application selects a relatively high vacuum degree of 1x10 -6 ~ 1x10 -7The collagen is soaked in Pa", and meanwhile, the collagen with a molecular weight of 5-300,000 is selected, and a proper concentration (1%-3%) of the collagen is controlled to control the proper viscosity of the collagen (if the concentration of the collagen is too low, the filling cannot be realized, and if the concentration is too high, the solution is too viscous, and the collagen cannot enter the pores), so that the molecular weight and the concentration of the collagen and the pores of the polylactic acid sponge support (the pores of the polylactic acid sponge support are determined by the molecular weight and the concentration of the polylactic acid solution) are optimally matched, the collagen molecules can be smoothly embedded in the internal pores of the polylactic acid under the condition of vacuum extraction, the internal pores of the polylactic acid sponge are filled, and the collagen is not only attached to the surface of the polylactic acid sponge support;

[0014] (3) Meanwhile, the smaller the molecular weight of the polylactic acid and the collagen is, the shorter the degradation period is, therefore, the polylactic acid and the collagen with a proper molecular weight are selected, and the composite biological support can also have a longer degradation period, so that the degradation rate of the composite biological support of the application is controlled at about 50% in five weeks, the degradation is avoided to be too fast to cause the pores to be generated too early and affect the protection effect on the residual ends of the ligament, and the degradation is also avoided to be too slow to cause the residual support to hinder the recovery of the function of the ligament;

[0015] (4) The application also realizes the stable formation of the double tyrosine bonds between the collagens and between the collagen and the polylactic acid by adopting the light-mediated photo-oxidation and reduction system, so that the collagen is solidified into glue in the pores of the polylactic acid, the porosity of the composite biological support is reduced, the mechanical strength of the composite biological support product is improved, and the service life of the composite biological support is prolonged;

[0016] (5) Before the freeze-drying in step (1), the sealing and slow pre-freezing are performed, so that the surface of the polylactic acid solution can be effectively prevented from being dehydrated too fast and a large number of ice crystals from being generated, the surface cracks are avoided, the stable filling of the collagen on the internal pores of the polylactic acid sponge support is affected, and the flatness of the product surface is affected, and the product is affected to be in contact with the surface of the ligament;

[0017] (6) The low porosity (below 65%) composite biological support is finally prepared, when the composite biological support is used to wrap the damaged position of the anterior cruciate ligament and is sutured with the anterior cruciate ligament, the composite biological support can isolate the synovial fluid and enzymes in the joint cavity outside the biological support, effectively protects the residual ends of the ligament, prevents the residual ends of the ligament from being attacked by plasmin and synovial fluid during the healing period, and realizes the functions of effectively reducing postoperative arthritis and promoting the rapid repair of the anterior cruciate ligament.

[0018] Further, the composite biological scaffold is in a solid cylindrical shape. For the anterior cruciate ligament rupture distance difference, the solid cylindrical composite biological scaffold can be connected between the two residual ends of the anterior cruciate ligament and sutured in situ between the composite biological scaffold and the anterior cruciate ligament. The composite biological scaffold can also be in a hollow cylindrical shape. For the rupture wound flat, the hollow cylindrical shape is directly used. Moreover, since the scaffold body is in a cylindrical or tubular shape, the corresponding polylactic acid sponge scaffold can also be in a cylindrical or tubular shape, at this time, the adhesion between the mold and the product is not easy to occur, which is beneficial to demolding, and at the same time, since the product edge and the bottom are relatively flat, the yield is improved.

[0019] Further, the concentration of transition metal complex ruthenium (Ru) in the ruthenium-peroxy disulfate sodium solution in step (2) is 0.01% to 0.1%, and the concentration of peroxy disulfate sodium is 0.01% to 1%. The ruthenium-peroxy disulfate sodium solution is mixed with the collagen solution at a volume ratio of 1:20 to 1:5.

[0020] Further, the visible light irradiation condition of step (3) is that the wavelength of visible light is 400-450 nm, the light intensity is 10-50 mW·cm −2 , and the irradiation time is 30 s-3 min.

[0021] Further, the soaking temperature of step (3) is controlled at 0-37℃ to maintain the activity of the protein.

[0022] Further, the mixed solution of step (2) is added with one or more than one bioactive substance. The bioactive substance is any one or more than one of growth factor, stem cell, platelet rich plasma (PRP), and drug; wherein the growth factor includes any one or more than one of epidermal growth factor (EGF), fibroblast growth factor (FGF), growth differentiation factor (GDF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), or transforming growth factor-beta (TGF-beta), and mechano growth factor E peptide; the stem cell includes any one or more than one of bone marrow mesenchymal stem cell (BMSC), adipose-derived mesenchymal stem cell (MSCs), and neural stem cell (NSCs); and the drug includes any one or more than one of tranexamic acid, celecoxib, indomethacin, glucosamine, and antibiotic drug. With the continuous degradation of the composite biological scaffold of the application, the active ingredients inside the scaffold are slowly released and continuously promote the growth of cells in the ligament, and finally effectively reduce postoperative arthritis and further promote the rapid repair function of the anterior cruciate ligament. The composite biological scaffold prepared in the application can be used for different patients in different situations, for example, for patients with high postoperative exercise requirements, stem cells and growth factors for promoting nerve repair can be compounded in the composite biological scaffold to promote the growth of nerve cells; for patients with low immunity, autologous PRP can be compounded in the composite biological scaffold to release various growth factors to promote repair; for patients with arthritis or other diseases, corresponding drugs can be compounded in the composite biological scaffold; therefore, the composite biological scaffold of the application can more flexibly promote the rapid and excellent repair of the anterior cruciate ligament, and maximize the recovery of the original limb function of the patient. Further, the concentration of the growth factor solution is preferably between 0.001 mg / L and 1 mg / L. The density of the stem cells is preferably between 25 million and 150 million cells / mL −1 . The platelet enrichment concentration of the platelet rich plasma is preferably between 4 and 8 times. The solution concentration of the drug is preferably between 0.5 and 1 mmol / L.

[0023] Further, in step (1), the polylactic acid solution is pre-cooled at -4℃ for 12-24 hours before being transferred to -20℃ for freezing for 12-24 hours to complete the slow pre-freezing.

[0024] In step (1), the mold is preferably a mold made of polyfluoroethylene material. Under the same conditions, compared with an iron mold or a glass mold, the polylactic acid sponge scaffold made of a mold made of polyfluoroethylene material has larger pores, which is beneficial for the filling of collagen in the subsequent process and is more conducive to wound healing.

[0025] Further, the organic solvent in step (1) is selected from any one of 4-dioxane, hexafluoroisopropanol, and trichloromethane.

[0026] Further, the alkali solution in step (2) is usually selected from any one of sodium hydroxide solution, potassium hydroxide solution.

[0027] The second object of the present application is to provide a composite biological scaffold for promoting anterior cruciate ligament repair, which is prepared by the preparation method of the composite biological scaffold for promoting anterior cruciate ligament repair according to the first object of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is a diagram of a ruptured anterior cruciate ligament;

[0029] Figure 2 is a schematic diagram of the use state of the composite biological scaffold in a solid cylindrical shape according to the present application;

[0030] Figure 3 is a schematic diagram of the use state of the composite biological scaffold in a hollow cylindrical shape according to the present application. DETAILED DESCRIPTION

[0031] The embodiments of the preparation method of the composite biological scaffold for promoting anterior cruciate ligament repair and the product thereof according to the present application will be described below with reference to the accompanying drawings.

[0032] A preparation method of a composite biological scaffold for promoting anterior cruciate ligament repair, comprising the following steps:

[0033] (1) Dissolve left-handed polylactic acid (PLLA) or polylactic acid-glycolic acid copolymer (PLGA) with a molecular weight of 250-500,000 in an organic solvent to obtain a polylactic acid solution with a concentration of 2%-5%, pour the obtained polylactic acid solution into a mold, vacuum degassing, then seal, slowly pre-freeze and freeze-dry to obtain a polylactic acid sponge scaffold;

[0034] (2) Dissolve type I or type III collagen (either natural animal source or recombinant) with a molecular weight of 5-30,000 in dilute acetic acid to obtain a collagen solution with a concentration of 1%-3%; dissolve a transition metal complex ruthenium (Ru) and sodium peroxodisulfate together in deionized water to obtain a ruthenium-sodium peroxodisulfate solution; add 1 mol / L sodium hydroxide solution dropwise to the collagen solution to adjust the pH value, to obtain a collagen solution with a pH value of 6-8; and mix the ruthenium-sodium peroxodisulfate solution with the collagen solution with a pH value of 6-8, stir uniformly to obtain a mixed solution;

[0035] (3) Soak the polylactic acid sponge scaffold obtained in step (1) in the mixed solution obtained in step (2), the mixed solution completely submerges the polylactic acid sponge scaffold, soak for 3-12 hours under vacuum condition, the vacuum degree is controlled at 1×10 -6 ~1×10 -7 Pa;

[0036] (4) taking out the polylactic acid sponge scaffold soaked in step (2), irradiating with visible light to perform in-situ crosslinking, to obtain a composite biological scaffold.

[0037] The present inventors investigated the effects of the polylactic acid solution concentration and the collagen solution concentration on the anti-permeation performance of the final composite biological scaffold product and the repair effect on the anterior cruciate ligament in mice during the experiment, and at the same time, used the simple polylactic acid sponge scaffold prepared by only performing step (1) of the present application as Comparative Example 1, used the simple photo-crosslinked collagen scaffold prepared by only performing steps (2) and (4) of the present application as Comparative Example 2, and used the polylactic acid / collagen composite biological scaffold prepared by only performing steps (1) to (3) of the present application without photo-crosslinking as Comparative Example 3, and the technical parameters corresponding to each example and comparative example are shown in Table 1.

[0038] Table 1

[0039]

[0040] The present inventors investigated the pore sealing, anti-slip liquid and enzyme permeation performance, and wet adhesion performance of the composite biological scaffold samples prepared in each example and comparative example. The specific test process is as follows:

[0041] Pore sealing: the scaffold porosity was tested by ethanol displacement method to investigate the void filling and sealing condition of the scaffold. First, the freeze-dried scaffold was weighed, and the weight was recorded as W0. Then, anhydrous ethanol was filled into a brown bottle, and the total weight was recorded as W1. Then, the freeze-dried scaffold was placed in the brown bottle, and ultrasonic degassing was performed to make the ethanol fill the scaffold, and then the ethanol was filled into the brown bottle again, and the total weight was recorded as W2. The scaffold in the brown bottle was quickly taken out, the remaining ethanol and the brown bottle were weighed and recorded as W3. The porosity calculation formula: porosity (%) = (W2-W3-W0) / (W1-W3) x 100%.

[0042] Anti-slip liquid and enzyme permeation performance: 10% BSA solution was used to simulate the anti-slip liquid and enzymes in the joint. First, the freeze-dried scaffold was weighed, and the weight was recorded as W0, and then it was soaked in 10% BSA solution. After soaking for 24 hours, the scaffold was taken out, freeze-dried, and the total weight was recorded as W1. Simulated joint fluid permeation amount: permeation amount (%) = (W1-W0) / W0 x 100%.

[0043] Wet adhesion property: The composite biological scaffold prepared was subjected to adhesion strength test by pig skin lap shear test. A fresh pig skin was subjected to fat removal and shaving treatment, and then the treated pig skin was cut into a rectangle of 10 mm x 30 mm, washed with water, taken out of the water and used directly without drying, the composite scaffold was pasted between two pieces of pig skin with a bonding area of 10 mm x 15 mm, then the adhesive area was clamped and placed at room temperature for 2 hours, a universal material testing machine (Instron 5567) was used to perform tensile test at a speed of 10 mm / min, and the calculation method of adhesion strength (Pa = N / m 2 ) was to divide the maximum force (N) by the bonding area (m 2 ).

[0044] Table 2

[0045]

[0046] As can be seen from the test results in Table 1 and Table 2, in the embodiments of the present application, the porosities of Examples 2 and 4 are significantly lower than those of other examples, indicating that the structures of the two groups are the most dense, because the collagen has a high molecular weight and is soaked for a long time, which can better form a dense structure scaffold. In addition, the porosities of Examples 5 and 6 are relatively large, mainly because the recombinant collagen has a relatively low molecular weight. By comparing Example 2 with Comparative Examples 1 and 2, it can be seen that a low or high concentration of collagen is not conducive to the filling of the pores of the polylactic acid sponge scaffold by collagen, and the porosity and joint fluid permeation amount of the composite biological scaffold sample prepared will increase, which is not conducive to the protection of the ligament stump. By comparing Example 2 with Comparative Example 3, it can be seen that a high concentration of polylactic acid will result in a polylactic acid sponge scaffold that is too dense, which is also not conducive to the filling of the pores by collagen, so the porosity and joint fluid permeation amount of the composite biological scaffold sample prepared are also significantly higher than those of the present application. Comparative Example 4 (a pure polylactic acid sponge scaffold) has the highest porosity, and the porosities of Comparative Examples 5 (a pure photo-crosslinked collagen scaffold) and 6 (a polylactic acid / collagen composite biological scaffold without photo-crosslinking) are also significantly higher than those of each embodiment of the present application, indicating that the scaffold structures of Comparative Examples 4 to 6 are relatively loose. The change trend of the permeation amount of the simulated joint fluid is similar to the porosity. Because a dense structure (low porosity) can effectively block the permeation of liquid. In addition, the wet adhesion property of Comparative Example 4 (a pure polylactic acid sponge scaffold) is the lowest, and the addition of collagen can endow the scaffold with certain wet adhesion property, which is conducive to the close adhesion of the scaffold to the ligament.

[0047] Since the porosities of the composite biological scaffold samples of the above Comparative Examples 1 to 3 are relatively high, the mechanical property tests are abandoned. Therefore, the present inventors only test and compare the mechanical properties (including the burst pressure, the storage modulus G', the compressive stress, the tensile displacement and the tensile maximum load) of the biological scaffold samples obtained from the above Examples 1 to 6 and Comparative Examples 4 to 6. The mechanical property test results of the above Examples 1 to 6 and Comparative Examples 4 to 6 are shown in Table 3. The specific test process is as follows:

[0048] Burst pressure: First, fresh pigskin is cut into a rectangle of 20 mm x 50 mm, and the pigskin is adhered to a plastic hose with cyanoacrylate glue, and a circular notch with a diameter of 3 mm is made on the pigskin and the hose with a needle. Then, the collagen-soaked scaffold is fixed at the notch, and then it is crosslinked into glue under light. The hydrogel is covered with gauze soaked in saline to prevent it from drying. One end of the hose is connected to a syringe pump, and the other end is connected to a digital pressure gauge. At the predetermined time (15 min and 120 min), the covering gauze is removed, and then PBS is injected into the syringe at a speed of 150 mL / h. At the same time, the burst pressure of the hydrogel is recorded.

[0049] Storage modulus: The storage modulus (G') of the hydrogel is obtained by performing an oscillation frequency sweep test with a rotational rheometer at 1% stress, 0.1 to 100 rad / s oscillation frequency and 25°C.

[0050] Compressive stress: The compression test is performed by uniaxial tension test in a Stable Micro Systems texture analyzer (TA-XT plus, UK). The measurement mode of the rotary texture analyzer is compression, and the test is performed at a speed of 20 mm / min under a strain of 80%.

[0051] Tensile displacement and tensile maximum load: The tensile mechanical property test is performed with a universal testing machine (LLOYD LR100K, CN). After the two end clamps are fixed, the tensile test is performed at a speed of 15 mm / min to obtain the maximum breaking load and tensile displacement of the scaffold.

[0052] Table 3

[0053]

[0054] As can be seen from the test results in Table 3, the various examples of the present application are significantly superior to Comparative Examples 4 to 6 in various indicators. The main reason is that the mechanical properties and fatigue resistance of the scaffold are improved after the collagen is compounded with polylactic acid. Higher mechanical properties are beneficial to the scaffold to resist certain external physical stress, so it is more suitable for application in the joint part.

[0055] Meanwhile, the present inventors also investigated the in-vitro degradation time and biocompatibility of the composite biological scaffold obtained in the above examples and comparative examples, and the test data are shown in Table 4 below. The specific test process is as follows:

[0056] In-vitro degradation: the prepared composite scaffold was weighed and placed in 6 500 mL beakers, 250 mL of phosphate buffer (PBS, pH = 7.4) and 2 × 10 5 U, and placed in a 37°C incubator. At 5 weeks, one sample was taken out, washed with deionized water, vacuum dried, and weighed, and the in-vitro degradation rate of the sample was the mass loss rate before and after the sample experiment. The experiment was repeated 3 times, and the average value of the experimental data was taken as the result.

[0057] Biocompatibility: (1) cell dead and live staining: a cylindrical hydrogel with a diameter of 10 mm was prepared, the hydrogel was soaked in PBS buffer and irradiated under ultraviolet for 24 h before the experiment, then the hydrogel was placed in a 24-well plate, and 50 μL of 3T3 cell suspension with a density of 4 × 105 cell / mL was inoculated on the hydrogel, and after 72 h of co-culture, the 3T3 cells were stained with live / dead cell staining solution, and the growth of the cells was observed under a fluorescence microscope. (2) Cell toxicity test: the culture solution and hydrogel cultured for 72 h in the well plate were aspirated, 200 μL of 0.05 mg / mL MTT solution was added to each well, and the plate was incubated in a 37°C constant temperature incubator containing 5% CO2 for 4 h. The MTT solution was aspirated, 200 μL of DMSO was added, and the plate was incubated in a 37°C constant temperature shaking incubator for 15 min, and then the plate was dispensed into a 96-well plate, and the absorbance value was tested at 490 nm wavelength using an enzyme-labeled instrument, and the relative proliferation rate of the cells was calculated.

[0058] Table 4

[0059]

[0060] As can be seen from the test results in Table 4, the polylactic acid scaffold of Comparative Example 4 degraded by 20.5% in five weeks, because the polylactic acid itself has a long degradation period, and the collagen of Comparative Example 5 completely degrades in five weeks because of the fast degradation time. However, too fast degradation of the material will lead to difficulty in playing the function of the scaffold and the function of promoting cell growth, and at the same time, too slow degradation will affect the regeneration of the ligament and the recovery of the function. The degradation period of the composite scaffold prepared in the examples of the present application can match the repair period of the ligament, which is beneficial to the growth of the ligament and the recovery of the joint function. At the same time, the collagen has excellent cell compatibility, so the addition of the collagen can improve the biocompatibility of the polylactic acid.

[0061] Of course, the alkali solution used for adjusting the collagen solution in the preparation method of the composite biological scaffold of the present application is not limited to sodium hydroxide solution, but can also be potassium hydroxide solution; and the concentration of the alkali solution is not limited to 1 mol / L, and the concentration can be adjusted as needed.

[0062] The organic solvent in step (1) is selected from any one of 4-dioxane, hexafluoroisopropanol, and trichloromethane.

[0063] The concentration of the transition metal complex ruthenium (Ru) in the ruthenium-sodium peroxodisulfate solution in step (2) is 0.01% to 0.1%, and the concentration of sodium peroxodisulfate is 0.01% to 1%.

[0064] The visible light irradiation condition in step (3) is: visible light wavelength 400-450 nm, light intensity 30 mW·cm −2 , irradiation time 30 s to 3 min. Among them, the volume ratio of the ruthenium-sodium peroxodisulfate solution to the collagen solution is controlled at 1:20 to 5, which is not limited to 1:10 in the examples.

[0065] Further, in step (1), the polylactic acid solution is slowly pre-frozen by first pre-cooling the polylactic acid solution at -4℃ for 12-24 h, and then freezing it at -20℃ for 12-24 h.

[0066] The mold in step (1) is preferably a mold made of polyfluoroethylene material. Under the same conditions, compared with iron molds or glass molds, the polyfluoroethylene material mold has larger pores, which is beneficial for subsequent collagen filling and is more conducive to wound healing.

[0067] Further, the mixed solution of step (2) is added with one or more than one bioactive substance. The bioactive substance is any one or more than one of growth factor, stem cell, platelet rich plasma (PRP), and medicine; wherein the growth factor includes any one or more than one of epidermal growth factor (EGF), fibroblast growth factor (FGF), growth differentiation factor (GDF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), and mechano growth factor E peptide; the stem cell includes any one or more than one of bone marrow mesenchymal stem cell (BMSC), adipose-derived mesenchymal stem cell (MSCs), and neural stem cell (NSCs); and the medicine includes any one or more than one of tranexamic acid, celecoxib, indomethacin, glucosamine, and antibiotic drug. The composite biological scaffold prepared by the present application can be used for different patients in different situations, and different bioactive substances can be compounded on the composite biological scaffold to more flexibly promote the rapid and excellent repair of the anterior cruciate ligament and maximize the recovery of the original limb function of the patient. Further, the concentration of the growth factor solution is preferably between 0.001 and 1 mg / L. The density of the stem cell is preferably between 25 and 150 million cells / mL. −1 The platelet enrichment concentration of the platelet rich plasma is preferably between 4 and 8 times. The solution concentration of the medicine is preferably between 0.5 and 1 mmol / L.

[0068] Further, the composite biological scaffold 100 is in a solid cylindrical shape (as shown in Figure 2 For the anterior cruciate ligament 200 with a large difference in fracture distance, the solid cylindrical composite biological scaffold 100 can be connected between the two residual ends of the anterior cruciate ligament 200 and sutured in situ between the composite biological scaffold 100 and the anterior cruciate ligament 200. Further, the composite biological scaffold 100 is in a hollow cylindrical shape (as shown in Figure 3 For the flat fracture wound, the hollow cylindrical form is directly used. Since the composite biological scaffold 100 is in a cylindrical shape, the corresponding polylactic acid sponge scaffold can also be in a cylindrical or tubular shape, and the mold and the product are not easy to adhere to each other, which is beneficial to demolding. At the same time, since the edges and the bottom of the product are relatively flat, the yield is improved.

[0069] The present application can be further deduced or replaced without departing from the concept of the present application, and all the simple deductions or replacements should be regarded as falling within the protection scope of the present application.

Claims

1. A method for preparing a composite biological scaffold that promotes anterior cruciate ligament repair, characterized in that, Includes the following steps: (1) Dissolve L-polylactic acid or polylactic acid-glycolic acid copolymer with a molecular weight of 250,000 to 500,000 in an organic solvent to obtain a first solution with a concentration of 2% to 5%. Pour the obtained first solution into a mold, remove bubbles under vacuum, seal it first, and then slowly pre-freeze and freeze dry to obtain a sponge scaffold. (2) Dissolve type I or type III collagen with a molecular weight of 50,000 to 300,000 in dilute acetic acid to obtain a collagen solution with a concentration of 1% to 3%; dissolve ruthenium, a transition metal complex, and sodium persulfate together in deionized water to obtain a ruthenium-sodium persulfate solution; add alkaline solution to the collagen solution to adjust its pH value to obtain a collagen solution with a pH of 6 to 8; The ruthenium-sodium persulfate solution obtained in this step is mixed with a collagen solution with pH 6-8, and stirred until homogeneous to obtain a mixed solution; (3) Immerse the sponge support obtained in step (1) in the mixed solution obtained in step (2), ensuring that the mixed solution completely submerges the sponge support, and immerse it under vacuum for 3 to 12 hours, with the vacuum degree controlled at 1×10⁻⁶. -6 ~1×10 -7 Pa; (4) Take out the sponge scaffold soaked in step (3) and perform in-situ cross-linking by visible light irradiation to obtain a composite biological scaffold.

2. The method for preparing the composite biological scaffold for promoting anterior cruciate ligament repair according to claim 1, characterized in that: The composite biological scaffold is in the form of a solid cylinder or a hollow tube.

3. The method for preparing the composite biological scaffold for promoting anterior cruciate ligament repair according to claim 1, characterized in that: In step (2), the concentration of the transition metal complex ruthenium in the ruthenium-sodium persulfate solution is 0.01%–0.1%, and the concentration of sodium persulfate is 0.01%–1%. The ruthenium-sodium persulfate solution is mixed with the collagen solution at a volume ratio of 1:20–1:

5. The visible light irradiation conditions in step (3) are: visible light wavelength 400–450 nm, light intensity 10–50 mW·cm. -2 Irradiation time: 30s to 3min.

4. The method for preparing the composite biological scaffold for promoting anterior cruciate ligament repair according to claim 1, characterized in that: The soaking temperature in step (3) is controlled between 0 and 37°C.

5. The method for preparing the composite biological scaffold for promoting anterior cruciate ligament repair according to claim 1, characterized in that: In step (2), one or more bioactive substances are added to the mixed solution; the bioactive substances are any one or more of growth factors, stem cells, platelet-rich plasma, and drugs; wherein, the growth factors include any one or more of epidermal growth factor, fibroblast growth factor, growth differentiation factor, insulin-like growth factor, platelet-derived growth factor, transforming growth factor-β, and mechanical growth factor E peptide; the stem cells include any one or more of bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, and neural stem cells; and the drugs include any one or more of tranexamic acid, celecoxib, indomethacin, glucosamine, and antibiotics.

6. The method for preparing the composite biological scaffold for promoting anterior cruciate ligament repair according to claim 5, characterized in that: The concentration of the growth factor solution is between 0.001 mg / L and 1 mg / L; the density of stem cells is 25 million to 15 million cells / mL. −1 The platelet enrichment concentration in platelet-rich plasma is between 4 and 8 times; the drug solution concentration is 0.5 to 1 mmol / L.

7. The method for preparing the composite biological scaffold for promoting anterior cruciate ligament repair according to claim 1, characterized in that: In step (1), when the first solution is slowly pre-frozen, it is first placed at -4°C for 12 to 24 hours for pre-cooling, and then transferred to -20°C for freezing for 12 to 24 hours to complete the slow pre-freezing.

8. The method for preparing the composite biological scaffold for promoting anterior cruciate ligament repair according to claim 1, characterized in that: The mold mentioned in step (1) is made of polyvinyl fluoride.

9. The method for preparing the composite biological scaffold for promoting anterior cruciate ligament repair according to claim 1, characterized in that: The organic solvent in step (1) is selected from any one of 4-dioxane, hexafluoroisopropanol, and chloroform; the alkaline solution in step (2) is selected from any one of sodium hydroxide solution and potassium hydroxide solution.

10. The composite biological scaffold prepared by the method for preparing the composite biological scaffold for promoting anterior cruciate ligament repair according to any one of claims 1 to 9.

Images