Lemon salt modified poly (lactic-co-glycolic acid) bioactive scaffold and preparation method thereof
By introducing basic fragments into poly(1,8-octanediol) and citrate-modified poly(lactic acid lactide) bioactive scaffolds, the problem of lack of continuous regulation in the treatment of osteonecrosis, bone defects and nonunion of existing scaffolds is solved, and bone tissue regeneration and self-repair are achieved.
Patent Information
- Application Number
- CN202511018369.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-23
- Publication Date
- 2025-10-17
AI Technical Summary
Existing scaffolds lack the ability to continuously regulate bone tissue regeneration when treating bone necrosis, bone defects and nonunion, resulting in poor repair effects.
By introducing the basic fragment N,N'-bis(2-hydroxyethyl)piperazine into a biodegradable elastomer of polycitrate-1,8-octanediol and preparing a porous scaffold using 3D printing technology, combined with a citrate-modified poly(lactic acid) lactide bioactive scaffold, the slow release of regulatory factors by metal ions stimulates osteogenic differentiation of bone marrow mesenchymal stem cells and inhibits inflammation, promoting angiogenesis.
It enhanced the adhesion and proliferation of rabbit bone marrow mesenchymal stem cells, promoted bone tissue regeneration, and improved the self-repair effect of osteonecrosis and nonunion.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of high polymer materials, in particular to a citrate-modified poly(lactide-co-glycolide) bioactive scaffold and a preparation method thereof. BACKGROUND
[0002] Because the adverse microenvironment of bone necrosis, bone defect and nonunion hinders the regeneration and self-repair of bone tissue, autologous iliac bone cell transplantation and allogeneic bone transplantation are usually used in clinic to treat bone defects, bone necrosis and nonunion, but they have problems such as limited source of material. The current research filling scaffold is only prepared by simply adsorbing the scaffold alone or with pro-angiogenic factors (VEGF) and bone morphogenetic proteins (BMP), which lacks the ability to slowly and continuously release regulatory factors, and it is difficult to achieve the promotion of bone tissue regeneration after implantation into the body, thereby affecting the self-repair of bone necrosis, bone defect and nonunion, resulting in poor repair effect.
[0003] A porous scaffold for treating bone defect animal models is prepared by introducing basic fragment N,N'-bis(2-hydroxyethyl)piperazine (BHEp) into the biodegradable elastomer of poly(lactic acid-co-glycolic acid)-1,8-octanediol and using 3D printing technology. Compared with the control group, the scaffold of the experimental group enhances the adhesion and proliferation of rabbit bone marrow mesenchymal stem cells and the expression of osteogenesis-related genes.
[0004] The scaffold prepared by the prior art only provides a filling material for bone necrosis, bone defect or nonunion, and achieves the purpose of facilitating cell ingrowth into the existing scaffold without having a sustained regulatory effect. The scaffold prepared by the prior art cannot improve the regeneration of bone tissue from the microenvironment, and thus cannot completely treat bone diseases such as bone necrosis, bone defect and nonunion.
[0005] Therefore, a bioactive scaffold with a sustained microenvironment regulatory effect is particularly important for treating bone necrosis, bone defect and nonunion diseases, and developing a preparation method thereof is a problem to be solved by those skilled in the art. SUMMARY
[0006] Therefore, a bioactive scaffold with a sustained microenvironment regulatory effect is particularly important for treating bone necrosis, bone defect and nonunion diseases, and developing a preparation method thereof is a problem to be solved by those skilled in the art.
[0007] The present application provides a preparation method of a citrate-modified poly(lactide-co-glycolide) bioactive scaffold, which comprises:
[0008] polymerizing low molecular weight polyglycolide with a citrate compound to obtain a polyglycolide prepolymer;
[0009] mixing the polyglycolide prepolymer with a porogen, and then performing a thermal crosslinking reaction, water washing and freeze-drying treatment to obtain a citrate-modified polyglycolide bioactive scaffold.
[0010] The application first mixes and reacts lactide, glycolide and a co-initiator to obtain low molecular weight polyglycolide. In some specific embodiments, the co-initiator includes but is not limited to one or more of 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, 1,7-heptanediol or 1,8-octanediol, and the application does not have special requirements for the selection of the co-initiator. In some specific embodiments, the reaction is performed in the presence of a solvent, which includes but is not limited to ethyl acetate, and the application does not have special requirements for the selection of the solvent. In some specific embodiments, the temperature of the reaction is 70-250°C, preferably 100-150°C, the time of the reaction is 1-120h, preferably 20-60h; the reaction is performed in the presence of a catalyst, which includes but is not limited to Sn(oct)2, and the application does not have special requirements for the selection of the catalyst. In some specific embodiments, after the reaction, the crude product is dissolved in chloroform, and then precipitated in ice anhydrous ether for 3 times to remove impurities, and finally a rotary evaporator is used to further remove residual solvents and impurities. In some specific embodiments, the molar ratio of lactide to glycolide is (1-5):1, the molar ratio of lactide to co-initiator is (1-3):1, and the mass percentage of the catalyst in the reaction raw material is (0.2-0.4)wt%, preferably 0.3wt%.
[0011] The application then carries out a polymerization reaction of the low molecular weight polyglycolide with a citrate-containing compound to obtain a polyglycolide prepolymer. In some specific embodiments, the number average molecular weight of the low molecular weight polyglycolide is 200 to 5000, preferably 700. In some specific embodiments, the molar ratio of the low molecular weight polyglycolide to the citrate-containing compound is 1:(0.5-2), preferably 1:1; the polymerization reaction is carried out in the presence of a polymerization catalyst, which includes but is not limited to one or more of stannous octoate (Sn(Oct)2), zinc octoate (Zn(Oct)2), or zinc oxide (ZnO), and the application does not have special requirements for the selection of the polymerization catalyst. In some specific embodiments, the molar ratio of the low molecular weight polyglycolide to the polymerization catalyst is 1:1. In some specific embodiments, the citrate-containing compound includes but is not limited to one or more of citric acid, magnesium citrate, manganese citrate, lithium citrate, strontium citrate, zinc citrate, or calcium citrate, and the application does not have special requirements for the selection of the citrate-containing compound. In some specific embodiments, the temperature of the polymerization reaction is 70°C to 250°C, preferably 120°C to 135°C; the time of the polymerization reaction is 10h to 120h, preferably 42h to 48h, and the reaction is stirred until it cannot be stirred.
[0012] The application then carries out a thermal crosslinking reaction of the polyglycolide prepolymer with a porogen, and a desalination treatment to obtain a citrate-modified polyglycolide bioactive scaffold. In some specific embodiments, the temperature of the thermal crosslinking reaction is 60°C to 250°C, preferably 90°C to 100°C, and the time of the thermal crosslinking reaction is 4h to 120h; the volume ratio of the polyglycolide prepolymer to the porogen is 1:(4-20). In some specific embodiments, the thermal crosslinking reaction is carried out in the presence of an organic solvent, which includes but is not limited to dioxane, and the application does not have special requirements for the selection of the organic solvent. In some specific embodiments, after the thermal crosslinking reaction, inverse molding and further thermal crosslinking are carried out, and then a desalination treatment is carried out, the temperature of the further thermal crosslinking is 100°C to 150°C, preferably 120°C, and the time of the further thermal crosslinking is 2 days to 6 days, preferably 4 days. In some specific embodiments, the time of the desalination treatment is 1 day to 14 days; the porogen includes but is not limited to one or more of sodium chloride, sodium tartrate, sodium citrate, or sucrose, and the application does not have special requirements for the selection of the porogen, and the particle size of the porogen is 25 mesh to 300 mesh.
[0013] The application also provides a citrate-modified polyglycolide bioactive scaffold, which is prepared according to the preparation method described above.
[0014] The present application changes the non-degradable octanediol in the prior art into polyglycolide-lactide which can control the degradation time according to the proportion of glycolide and lactide, citrate as a thermal crosslinking agent, and metal ions with regulatory cell function are doped in the polymerization reaction, so as to achieve the purpose of slow and continuous release of regulatory factors with the continuous degradation of the scaffold. BRIEF DESCRIPTION OF DRAWINGS
[0015] Figure 1 NMR spectrum of low molecular weight polyglycolide-lactide provided for Example 1 of the present application;
[0016] Figure 2 Mass spectrum of low molecular weight polyglycolide-lactide provided for Example 1 of the present application;
[0017] Figure 3 CCK-8 test chart of citrate modified polyglycolide-lactide bioactive scaffold provided for Examples 1, 2, 3, 5, and 6 of the present application;
[0018] Figure 4 Compression curve chart of citrate modified polyglycolide-lactide bioactive scaffold provided for Examples 1-7 of the present application;
[0019] Figure 5 Porosity chart of citrate modified polyglycolide-lactide bioactive scaffold provided for Examples 1-7 of the present application;
[0020] Figure 6 EDS scan chart of citrate modified polyglycolide-lactide bioactive scaffold provided for Example 1 of the present application;
[0021] Figure 7 Live and dead staining chart of citrate modified polyglycolide-lactide bioactive scaffold provided for Example 1 of the present application;
[0022] Figure 8 Rabbit bone necrosis model in vivo NMR chart of citrate modified polyglycolide-lactide bioactive scaffold provided for Example 1 of the present application;
[0023] Figure 9 Rabbit bone necrosis model in vivo CT chart of citrate modified polyglycolide-lactide bioactive scaffold provided for Example 1 of the present application. DETAILED DESCRIPTION
[0024] It should be understood that the expression "one or more of" includes each of the objects recited after the expression and various different combinations of two or more of the recited objects, individually, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.
[0025] The use of the terms "including", "containing", or "comprising" and variations thereof, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items not specifically listed, unless otherwise specified or limited by context.
[0026] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the application remains operable. Moreover, two or more steps or actions can be conducted simultaneously.
[0027] The use of any and all examples, or exemplary language herein, is intended merely to better illuminate the application and does not pose a limitation on the scope of the application unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the application.
[0028] Further, the numerical ranges and parameters setting forth the broadest scope of the application are approximations, and are used to encompass the range of values falling within the scope of the application. Unless otherwise specified, the numerical values are approximations only, and thus the numerical values disclosed are intended to be used only to convey a rough estimate of the numerical values to the skilled worker. Any numerical value, however, can be exactly or approximately the numerical value specified. Any numerical value can be expressed with a degree of error, for example, a range of error of ± 10%, ± 5%, ± 1%, or ± 0.5% of the stated value. Thus, any numerical value can be expressed with a degree of error that is appropriate for the circumstances.
[0029] The application provides a preparation method of a citrate-modified polyglycolide-lactide bioactive scaffold, comprising:
[0030] Polymerizing a low molecular weight polyglycolide with a citrate-containing compound to obtain a polyglycolide prepolymer;
[0031] Thermally cross-linking the polyglycolide prepolymer with a porogen, washing with water, and freeze-drying to obtain a citrate-modified polyglycolide-lactide bioactive scaffold.
[0032] The application changes the non-degradable octanediol in the prior art into polyglycolide-lactide as a raw material, which can control the degradation time according to the ratio of lactide and glycolide, uses citrate as a thermal cross-linking agent, and dopes metal ions with a cell regulation effect in the polymerization reaction, so as to achieve the purpose of slow and continuous release of regulatory factors with the continuous degradation of the scaffold.
[0033] The application will be further described in conjunction with the following examples. The protection scope of the application is not limited by the following examples.
[0034] Example 1
[0035] The embodiment provides a citrate-modified polyglycolide bioactive scaffold, and a preparation method of the citrate-modified polyglycolide bioactive scaffold.
[0036] The required reagents, such as 1,6-hexanediol, lactide, glycolide, catalyst Sn(oct)2, citric acid, hydroxyapatite and sodium chloride particles, were accurately weighed, and solvents such as dioxane, chloroform and diethyl ether were prepared. Among them, the amount of each reagent is as follows: 17.2956 g of lactide, 4.64 g of glycolide, 4.7268 g of 1,6-hexanediol and 0.08147 g of Sn(oct)2.
[0037] Preparation of experimental apparatus: 250 mL round-bottom flask, oil bath, magnetic rotor, rotary evaporator, oven, liquid nitrogen, freeze dryer, vacuum drying oven, rubber plug, analytical balance, screen and other experimental apparatus were prepared, and it was ensured that the apparatus could normally operate.
[0038] Preparation of low molecular weight polyglycolide:
[0039] Lactide and glycolide recrystallization: lactide and glycolide were placed in a 60°C water bath, anhydrous ethyl acetate was added until they were completely dissolved, then they were left to crystallize at room temperature, and the operation was repeated 3 times.
[0040] Preparation of reaction system: the bottle used for the reaction was baked to remove moisture, the recrystallized lactide, glycolide and 1,6-hexanediol were added to the bottle, the experimental apparatus was connected, and the air in the apparatus was removed by charging and discharging nitrogen 3 times.
[0041] Melting by heating: the temperature of the oil bath was set to 130°C to completely melt the lactide and glycolide.
[0042] Polymerization reaction: the bottle cap was opened to add the catalyst Sn(oct)2, the apparatus was sealed again and vacuumized, and the temperature was maintained for 48 h.
[0043] Product purification: after the reaction was completed, the crude product was dissolved in chloroform, and impurities were removed by precipitating 3 times in anhydrous diethyl ether, and finally the residual solvent and impurities were further removed by using a rotary evaporator.
[0044] Product characterization: the obtained product was subjected to nuclear magnetic and mass spectrometry analysis to characterize the molecular weight. The product is off-white in appearance, viscous and uniform in texture. The nuclear magnetic hydrogen spectrum of the low molecular weight polyglycolide provided by the embodiment is shown in Figure 1 , and the mass spectrum of the low molecular weight polyglycolide provided by the embodiment is shown in Figure 2 .
[0045] Prepolymerization of polyglycolide and CA:
[0046] Material addition: 6.9427 g of polyglycolide and 1.72 g of citric acid were added to a 250 mL round bottom flask.
[0047] Catalyst addition: Catalyst was added to the flask.
[0048] Reaction apparatus connection and heating: After connecting the vacuum pump, the oil bath temperature was set to 130°C for heating.
[0049] Reaction progress: The reaction was stopped when the magnetic rotor could not stir the reaction mixture after continuous vacuuming and heating for four days. The resulting product was brownish yellow, in a viscous and uniform state.
[0050] Preparation of the scaffold:
[0051] Prepolymer dissolution: The prepolymer of citric acid and polyglycolide (CA-PLGA) was dissolved in dioxane.
[0052] NaCl particle screening: NaCl particles were screened using a screen mesh, and particles with a particle size between 75 and 40 mesh (i.e., 200 μm-400 μm) were selected for use.
[0053] Particle addition and mixing: According to the designed porosity, the screened NaCl particles were accurately added according to the volume ratio of NaCl to CA-PLGA, and then stirred to fully mix.
[0054] First step of thermal crosslinking reaction: The uniformly mixed material was placed in an oven and reacted at 100°C for 4h for the second step of thermal crosslinking reaction.
[0055] Molding: The material after the second step of thermal crosslinking reaction was taken out and placed in a mold to prepare a uniform specification material and salt blend block.
[0056] Further thermal crosslinking: The standard material and salt blend block was placed in a vacuum drying oven and further thermal crosslinking was carried out at 120°C for 4 days until the scaffold color changed from white to yellow, and then removed.
[0057] Desalination treatment: The thermally crosslinked standard block was placed in water and desalination treatment was carried out under continuous stirring of the rotor for 4 days.
[0058] Freeze-drying treatment: The scaffold after salt elution was placed in a freeze dryer for freeze-drying treatment.
[0059] Storage: To prevent degradation of the scaffold, the prepared scaffold was stored in a -80°C refrigerator.
[0060] Example 2
[0061] This example provides a citrate-modified polyglycolide bioactive scaffold, which differs from Example 1 in that 1.72 g of citric acid is replaced with 1.548 g of citric acid and 0.1916 g of magnesium citrate.
[0062] Example 3
[0063] This example provides a citrate-modified polyglycolide bioactive scaffold, which differs from Example 1 in that 1.72 g of citric acid is replaced with 1.548 g of citric acid and 0.2195 g of manganese citrate.
[0064] Example 4
[0065] This example provides a citrate-modified polyglycolide bioactive scaffold, which differs from Example 1 in that 1.72 g of citric acid is replaced with 1.548 g of citric acid and 0.1868 g of lithium citrate.
[0066] Example 5
[0067] This example provides a citrate-modified polyglycolide bioactive scaffold, which differs from Example 1 in that 1.72 g of citric acid is replaced with 1.548 g of citric acid and 0.2472 g of strontium citrate.
[0068] Example 6
[0069] This example provides a citrate-modified polyglycolide bioactive scaffold, which differs from Example 1 in that 1.72 g of citric acid is replaced with 1.548 g of citric acid and 0.1699 g of zinc citrate.
[0070] Example 7
[0071] This example provides a citrate-modified polyglycolide bioactive scaffold, which differs from Example 1 in that 1.72 g of citric acid is replaced with 1.548 g of citric acid and 0.14774 g of calcium citrate.
[0072] The citrate-modified polyglycolide bioactive scaffolds provided in Examples 1-7 are subjected to performance testing, and the testing methods are as follows:
[0073] Cell proliferation rate: CCK-8 is used to detect cell proliferation rate. The absorbance of cell culture solution at a specific wavelength (450 nm) is measured by a microplate reader, and the absorbance is proportional to the amount of formazan, which can reflect the number of living cells. By comparing the absorbance at the original time point, the cell proliferation rate can be calculated;
[0074] Stent compression: A universal calibrator is used to test the stent compression performance. By applying vertical pressure to the sample, the sensor measures the pressure and sample deformation in real time. Based on Hooke's law and other related principles, the sample compression performance parameters are calculated from the collected data.
[0075] Porosity: Use a vacuum densitometer to test the porosity of the scaffold. Based on the Archimedean principle, the true density of the material (excluding the density of pores) is first determined, and then the apparent density (including the density of pores) is measured. The porosity of the material is calculated from the density data of the two.
[0076] EDS scanning: Based on the detected X-ray energy and the reference to the characteristic X-ray energy spectrum of known elements, the types and distribution of elements present in the sample can be determined, completing qualitative analysis. By detecting the relationship between the characteristic X-ray intensities and comparing them with the intensity of standard samples, quantitative analysis of the relative content of each element can be achieved.
[0077] Live-dead staining: The content and distribution of the two types of cells are demonstrated by the uptake of two specific dyes by dead cells and living cells respectively. It is used to determine the survival status of cells under the co-culture conditions of scaffolds and cells, and to prove the proliferation-promoting and induction properties of the scaffolds.
[0078] In vivo MRI of rabbit osteonecrosis model: By performing MRI on individual samples of rabbit femoral head necrosis model treatment group under anesthesia, the extent of femoral head necrosis, degree of bone marrow edema, and degree of femoral head collapse can be effectively evaluated;
[0079] In vivo CT of rabbit osteonecrosis model: By performing CT examination on individual samples of the rabbit femoral head necrosis model treatment group under anesthesia, the bone regeneration in the femoral head necrosis area can be evaluated.
[0080] The CCK-8 test results of the lemon salt modified poly (lactide-co-glycolide) bioactive scaffolds provided in Examples 1, 2, 3, 5, and 6 are shown in FIG. Figure 3 The compression curves of the lemon salt modified poly (lactide-co-glycolide) bioactive scaffolds provided in Examples 1-7 are shown in FIG. Figure 4 As shown; the porosity of the lemon salt modified poly (lactide) bioactive scaffold provided in Examples 1-7 is shown in FIG. Figure 5 As shown; the EDS scanning diagram of the lemon salt modified poly (lactide-co-glycolide) bioactive scaffold provided in Example 1 is as shown Figure 6 As shown; the live-dead staining diagram of the lemon salt modified poly (lactide-glycolide) bioactive scaffold provided in Example 1 is as shown Figure 7 As shown; the in vivo nuclear magnetic resonance image of the rabbit osteonecrosis model of the lemon salt modified poly (lactide-co-glycolide) bioactive scaffold provided in Example 1 is as shown Figure 8 As shown; the in vivo CT image of the rabbit osteonecrosis model of the lemon salt modified poly (lactide) bioactive scaffold provided in Example 1 is as shown Figure 9 shown.
[0081] From the test, we know that:
[0082] From the cell proliferation rate, we know that the scaffold has a proliferation effect on bone marrow mesenchymal stem cells.
[0083] From the compression test, we know that the compression performance of the scaffold reaches the strength of 1.2MPa.
[0084] From the porosity determination, we know that the porosity of the scaffold reaches more than 85%.
[0085] From the EDS scan, we know that the metal ions are uniformly distributed on the scaffold, confirming the successful material encapsulation.
[0086] From the live and dead staining, we know that the scaffold has an induction effect on bone marrow mesenchymal stem cells, and the cells spread and grow on the scaffold wall.
[0087] From the rabbit bone necrosis model in vivo nuclear magnetic resonance, we know that after the rabbit model is implanted with the scaffold for treatment, the bone marrow edema is reduced, the inflammation is controlled, and the bone necrosis site is improved.
[0088] From the rabbit bone necrosis model in vivo CT, we know that after the rabbit model is implanted with the scaffold for treatment, osteogenesis appears, indicating that the scaffold promotes new bone growth.
[0089] The above is only the preferred specific implementation of the present application, but the protection scope of the present application is not limited to this, any skilled person in the art can make equivalent replacement or change according to the technical scheme and application concept of the present application within the technical range disclosed by the present application, which should be covered in the protection scope of the present application.
Claims
1. A method for preparing a citric acid salt-modified poly (lactide-glycolide) bioactive scaffold, characterized in that: include: Polymerizing low molecular weight poly(lactide-glycol) with a compound containing a citrate radical to obtain a poly(lactide-glycol) prepolymer; The poly(lactic acid) glycolide prepolymer is mixed with a porogen, subjected to a thermal cross-linking reaction, washed with water and freeze-dried to obtain a citric salt-modified poly(lactic acid) glycolide bioactive scaffold.
2. The preparation method according to claim 1, characterized in that The number average molecular weight of the low molecular weight poly(lactide-glycolide) is 200 to 5000.
3. The preparation method according to claim 1, characterized in that The preparation method of the low molecular weight poly(lactide-glycolide) comprises: The lactide, glycolide and co-primer are mixed and reacted to obtain low molecular weight poly (lactide-glycolide); The molar ratio of the lactide to the glycolide is (1-5):1, and the molar ratio of the lactide to the co-primer is (1-3):
1.
4. The preparation method according to claim 3, characterized in that The co-primer includes one or more of 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, 1,7-heptanediol or 1,8-octanediol; The reaction temperature is 70° C. to 250° C., and the reaction time is 1 h to 120 h. The reaction is carried out in the presence of a catalyst, and the catalyst includes Sn(oct)2.
5. The preparation method according to claim 1, characterized in that The molar ratio of the low molecular weight poly(lactide-glycolide) to the citrate-containing compound is 1:(0.5-2); the polymerization reaction is carried out in the presence of a polymerization catalyst, and the polymerization catalyst includes one or more of stannous octoate, zinc octoate or zinc oxide.
6. The preparation method according to claim 1, characterized in that The citrate-containing compound includes one or more of citric acid, magnesium citrate, manganese citrate, lithium citrate, strontium citrate, zinc citrate or calcium citrate.
7. The preparation method according to claim 1, characterized in that The polymerization reaction temperature is 70° C. to 250° C.; the polymerization reaction time is 10 h to 120 h.
8. The preparation method according to claim 1, characterized in that The temperature of the thermal crosslinking reaction is 60° C. to 250° C., and the time of the thermal crosslinking reaction is 4 hours to 120 hours. The volume ratio of the poly(lactide-glycolide) prepolymer to the porogen is 1:(4-20).
9. The preparation method according to claim 1, characterized in that The desalination treatment time is 1 day to 14 days; the porogen includes one or more of sodium chloride, sodium tartrate, sodium citrate or sucrose; and the particle size of the porogen is 25 mesh to 300 mesh.
10. A citric acid salt modified poly (lactide-glycolide) bioactive scaffold, characterized in that: It is prepared according to the preparation method according to any one of claims 1 to 9.