Preparation method of cross-linking polymer and application of cross-linking polymer in adaptive DLP printing

Abstract

The invention provides a crosslinking polyfunctional polymer, the prepolymer system of which is high in reaction activity and low in viscosity, has certain flowability at room temperature, and is adaptive to digital light processing 3D printing (DLP). The preparation method of the cross-linking polymer comprises the following steps: carrying out ring-opening polymerization on caprolactone by using polyol as an initiator to prepare a polyfunctional polycaprolactone precursor with the molecular weight of 300Da-800Da, and introducing a propenyl compound to the molecular terminal of the polycaprolactone precursor to obtain a double-bond modified low-molecular-weight polycaprolactone reaction product. And carrying out primary centrifugation, filtration and ethyl acetate elution on the reaction product to obtain a transparent colorless solution, carrying out rotary evaporation to remove the solvent, fully washing with water, carrying out secondary centrifugation, and drying to obtain the polycaprolactone prepolymer. Through verification, the polycaprolactone prepolymer disclosed by the invention has the characteristics of biosafety, biodegradability, low viscosity flowability, high-strength mechanical elastic modulus and the like, and can adapt to DLP 3D printing to prepare medical instruments.

Description

Technical Field

[0001] The invention belongs to the field of biomaterials, and in particular relates to a low-viscosity biodegradable cross-linked polymer system suitable for DLP printing and a preparation method thereof. Background Art

[0002] DLP (Digital Light Processing) is a type of 3D printing technology, also known as digital light processing rapid prototyping technology. The basic principle is to use digital light sources to project layer by layer on the surface of liquid photosensitive resin in the form of surface light, and then solidify and shape the layers, and repeat this cycle until the final model is completed. DLP molding technology generally uses photosensitive resin as the printing material. Currently, the consumables for DLP printing on the market are mainly acrylic resins and silicone resins, but the potential toxicity of these photosensitive resin systems limits their application in the medical field.

[0003] At present, the biomaterials suitable for DLP bio-3D printing are basically photocurable hydrogel systems, such as methacryloyl gelatin GELMA hydrogel and polyethylene glycol PEGDA hydrogel, etc. However, the compression modulus of these photocurable hydrogels is below the MPa level, which is difficult to meet the requirements in some tissue repair environments with higher mechanical requirements.

[0004] Laura Elomaa et al. reported a technical solution for preparing light-cured PCL (doi:10.1016 / j.actbio.2011.06.039), which prepared PCL materials that can be heated, melted and then light-cured. However, they mentioned that the small molecular weight PCL prepared by their method is still dissolved in the system prepared by the reaction and cannot be purified by sedimentation separation. Therefore, during the printing process, it is necessary to heat the provided PCL material to above the melting point, and then cross-link it by light after the melt viscosity becomes low. Since 3D printing of PCL materials can be achieved by heating, melting, cooling and curing, there is no need to add light curing.

[0005] Existing implantable biomaterials suitable for DLP bio-3D printing cannot simultaneously meet the material's biosafety and high-strength mechanical elastic modulus. Therefore, materials that can meet the requirements of high strength, biodegradability, biosafety, and low-viscosity flowable photo-cross-linking are urgently needed for clinical applications. Summary of the invention

[0006] One object of the present invention is to provide a method for preparing a cross-linked polymer for use in manufacturing DLP biological 3D printing materials.

[0007] Another object of the present invention is to provide a method for preparing a cross-linked polymer, using polycaprolactone as the main polymer and performing double bond modification at both ends of the molecule to obtain a fluid material that is suitable for DLP printing.

[0008] Another object of the present invention is to provide a method for preparing a cross-linked polymer, using polycaprolactone as the main polymer and performing double bond modification at both ends of the molecule to obtain a material with biosafety and high-strength mechanical elastic modulus, which can be adapted to DLP biological 3D printing and used for tissue repair with higher mechanical requirements.

[0009] Another object of the present invention is to provide an application of the low molecular weight polycaprolactone modified by double bonding in DLP biological 3D printing.

[0010] The fifth object of the present invention is to provide a composition suitable for DLP biological 3D printing, which contains a low molecular weight polycaprolactone modified by double bonding.

[0011] A method for preparing double-bond modified low molecular weight polycaprolactone is as follows:

[0012] Using a polyol with a functionality n in the range of 2-6 as an initiator, adjusting the ratio of caprolactone to polyol (the content of caprolactone monomer is m times the hydroxyl equivalent of the polyol, 1≤m≤5) so that the molecular weight of the obtained polycaprolactone precursor is between 300Da and 800Da;

[0013] Using potassium carbonate or cesium carbonate as an acid-binding agent, an propylene compound is introduced into the molecular end of a polycaprolactone precursor to obtain a reaction product containing a low molecular weight polycaprolactone modified by double bonds;

[0014] After the reaction is completed, the reaction product is centrifuged for the first time (e.g., 500 g to 1000 g for 10 to 30 minutes, preferably 800 g for 30 minutes), filtered (e.g., filtered through a sand core funnel and diatomaceous earth), and ethyl acetate is used as an eluent to remove excess potassium carbonate to obtain a clear, transparent, colorless solution. The solvent is then removed by reduced pressure distillation (e.g., a rotary evaporator), and then fully washed with water (finally, the pH of the washing solution is made neutral, and no precipitation is generated when a sample is added with silver nitrate as the washing end point), and then centrifuged again and the resulting precipitated product is dried to obtain a polycaprolactone prepolymer.

[0015] The propenyl compounds suitable for the method of the present invention include, but are not limited to, acryloyl chloride or methacrylic anhydride.

[0016] The polyol initiating monomers suitable for the method of the present invention include, but are not limited to, ethylene glycol, 1,3-propylene glycol, trimethylolpropane (TMP), octanedetrol, and pentaerythritol.

[0017] In the method of the present invention, the speed of the second centrifugation is: 1000g-1500g, the time is 20-30 minutes, preferably 1200g, 30 minutes.

[0018] According to detection, the average molecular weight of the polycaprolactone prepolymer prepared by the present invention is 400-1000Da, such as 400Da, 500Da, 600Da, 700Da, 800Da, 900Da and 1000Da.

[0019] Through testing, the viscosity of the polycaprolactone prepolymer prepared by the present invention is 100-600 mpa·s, such as 100 mpa·s, 200 mpa·s, 300 mpa·s, 400 mpa·s, 500 mpa·s and 600 mpa·s.

[0020] According to the test, the mechanical elastic modulus of the polycaprolactone prepolymer prepared by the present invention is 1 to 100 MPa, such as 1 MPa, 10 MPa, 20 MPa, 30 MPa, 40 MPa, 50 MPa, 60 MPa, 70 MPa, 80 MPa, 90 MPa and 100 MPa. The functionalities of the prepared PCL prepolymers are different due to the different polyols used, and the difference in the final crosslinking density leads to different mechanical moduli of the obtained PCL. For example, by selecting a single polyol to prepare products with different mechanical moduli, or by mixing several polyols, products with different mechanical moduli can be provided on demand.

[0021] The present invention proposes a technical strategy of using water washing and centrifugation, which can not only remove the reaction aid, but also separate the prepared low molecular weight PCL, and avoids the use of excessive organic solvent for sedimentation purification in the reaction purification process in the reference (doi:10.1016 / j.actbio.2011.06.039).

[0022] Compared with the photocurable PCL prepared by Laura Elomaa et al., the polycaprolactone prepolymer prepared by the present invention has a smaller molecular weight photocurable PCL, is liquid at room temperature, and can be directly used for liquid DLP photocurable printing without melting.

[0023] Therefore, the polycaprolactone prepolymer prepared in the present invention has the characteristics of biosafety, biodegradability, low viscosity flowability and high strength mechanical elastic modulus, and is suitable for implantable medical devices prepared by DLP biological 3D printing.

[0024] The liquid polymer system adapted to DLP printing is composed of double-bonded modified low molecular weight polycaprolactone as the main substance, supplemented by active diluents (such as polyethylene glycol PEG, polyethylene glycol acrylate PEGDA, hydroxyethyl methacrylate HEMA and polyglycerol acrylate PPGME) and photoinitiators (such as but not limited to benzoin methyl ether, content 0.1wt% to 1wt%), etc. Among them, the content of double-bonded modified low molecular weight polycaprolactone is 30% to 90% by weight, preferably 60% to 80%. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 A schematic diagram of a route for preparing double-bond terminated polycaprolactone according to the present invention;

[0026] Figure 2 The hydrogen nuclear magnetic spectra of the polycaprolactone reaction precursor and the double-bond terminated polycaprolactone prepared by the present invention;

[0027] Figure 3 This is a graph showing the rheological test results of the polycaprolactone prepolymer prepared in the present invention;

[0028] Figure 4 The polycaprolactone prepolymer prepared by the present invention is used for DLP biological 3D printing product diagram;

[0029] Figure 5 The mechanical properties test results of PCL polymer produced by DLP 3D printing;

[0030] Figure 6 The figure shows the proliferation results of mesenchymal stem cells on the polycaprolactone scaffold prepared by DLP printing, where Figure A is a cytoskeleton image presented under a fluorescence microscope, and Figure B is a statistical graph of the proliferation data. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the embodiments, it should be understood by those skilled in the art that the technical solution of the invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

[0032] The various test methods used in the following examples of the present invention are specifically described as follows:

[0033] 1) Nuclear magnetic hydrogen spectrum testing and characterization methods

[0034] The prepared PCL prepolymer was dissolved in CDCl3 and placed on a 400 MHz nuclear magnetic hydrogen spectrum NMR (Bruker) for chemical shift spectrum characterization and analysis, with the scanning parameter set to 16 times.

[0035] 2) Molecular weight testing and characterization

[0036] The relative molecular weight of the prepared PCL prepolymer was tested by gel permeation chromatography, with tetrahydrofuran (THF) as the eluent at a flow rate of 1.0 ml / min and the temperature being constant at 35°.

[0037] 3) PCL prepolymer rheological properties test

[0038] The liquid viscosity was tested using a torque rheometer (Malvern) with a 60 mm vertebral plate (1° tilt angle) and the shear rate was adjusted to 0.1 to 1000 s. -1 .

[0039] 4) DLP Bio-3D Printing

[0040] An appropriate amount of the prepared PCL prepolymer was added to the orifice plate of a 3D printer (Cyberiad, Azure 550A), and light-curing 3D printing was performed by setting the light intensity to 30% and the curing time per layer to 200 ms.

[0041] 5) Mechanical properties testing and characterization methods

[0042] PCL polymer was mechanically characterized by a universal mechanical testing machine (Instron 5966,) to measure the strength and compression modulus of the material under compression. The sample was subjected to a compressive force perpendicular to its surface, and the load stress and compressive strain data were recorded, and the compression modulus was calculated.

[0043] 6) Extraction and culture of mesenchymal stem cells

[0044] Fresh mesenchymal stem cells were extracted from the bone marrow of 7-day-old SD rats. After culture and subculture, the second-generation mesenchymal stem cells were cultured at a concentration of 2×10 6 The density of cells was dropped on the surface of the printed scaffold, and finally the scaffold sample seeded with mesenchymal stem cells was placed in a cell culture incubator for culture.

[0045] Example 1 Preparation of polycaprolactone prepolymer

[0046] The preparation route of the polycaprolactone prepolymer of this embodiment is as follows Figure 1 As shown, trimethylolpropane (TMP) initiates the ring-opening polymerization of caprolactone (CL), and acryloyl chloride reacts to introduce photocrosslinkable groups.

[0047] Before the reaction preparation, the dichloromethane solvent was stirred and refluxed with calcium hydride or anhydrous calcium chloride under a high-purity argon atmosphere, and the solvent was stored and sealed in a cool and dry place for subsequent experiments. The acid-binding agent potassium carbonate was ground in a mortar in advance, placed in a 120°C oven to remove water, cooled to room temperature under vacuum conditions, sealed and placed in a cool and dry place for subsequent experiments.

[0048] The glass reactor is dried under vacuum at high temperature for half an hour to be fully dried, TMP and three times the equivalent of CL are added under argon protection, and after vacuum drying for half an hour, one ten-thousandth to one thousandth of a catalyst stannous octoate is added, and the reaction is carried out at 120-160°C for 2 hours to obtain a multifunctional polycaprolactone precursor (polycaprolactone polyol).

[0049] Add dehydrated dichloromethane to the polycaprolactone polyol to completely dissolve the polycaprolactone polyol. Separately, 3 times the equivalent (3 times the equivalent relative to the hydroxyl content of the polycaprolactone polyol) of acryloyl chloride is dissolved in dichloromethane and slowly added dropwise to the reaction system using a syringe pump. Since a large amount of heat is released during the reaction process, the entire reaction needs to be kept under ice bath conditions, and 2 times the equivalent (2 times the equivalent relative to the acryloyl chloride content) of anhydrous potassium carbonate is added to the reaction bottle to remove the acidic product produced during the reaction. After the addition of acryloyl chloride is completed, the reaction is continued for 48 hours to obtain a reaction product containing a low molecular weight polycaprolactone with double bonds modified at both ends.

[0050] After the reaction is completed, the product is first centrifuged (800g, 30 minutes) to remove most of the insoluble salts, and the mixed liquid is filtered with diatomaceous earth, and ethyl acetate is used as an eluent to remove excess potassium carbonate to obtain a clear, transparent, colorless solution. After the solvent is removed by reduced pressure distillation, some water-soluble by-products are fully washed with water, and the product is centrifuged again (1000g~1500g, time is 20~30 minutes) to remove water and then separated and dried to obtain a polycaprolactone prepolymer.

[0051] The hydrogen spectra of polycaprolactone before and after double bond termination were characterized by 400 MHz nuclear magnetic hydrogen spectrum (Bruker, internal standard CDCl3). Figure 2 As shown. Figure 2 It can be seen that the polycaprolactone prepolymer prepared in this embodiment has the modification characteristics of unsaturated double bonds. Characterized by NMR peak integration and GPC, the average molecular weight of the polycaprolactone prepolymer prepared in the present invention is 500 Da.

[0052] Example 2 Material Properties Characterization

[0053] The obtained polycaprolactone prepolymer was characterized by a torque rheometer and showed low viscosity and good fluidity, and its shear viscosity decreased slightly with the increase of shear rate. Figure 3 shown.

[0054] Example 3DLP Bio-3D Printing

[0055] The double-bonded modified low molecular weight polycaprolactone prepared in this embodiment is used as the main substance, and an active diluent (such as hydroxyethyl methacrylate) and a photoinitiator are used to form a composition suitable for DLP printing. The composition is in a liquid state, and the content of the double-bonded modified low molecular weight polycaprolactone is 30% to 90% by weight, especially 60% to 80%.

[0056] The light-cured microneedle products (size 2 cm × 2 cm) made by 3D printer (Cyberiad, Azure 550A) were Figure 4 The mechanical properties of the prepared PCL polymer were characterized by a universal mechanical testing machine (Instron 5966, ). Figure 5 As shown in the figure, its compression modulus is much higher than the KPa level of general photocurable hydrogel. Different polyols can be used to obtain polycaprolactone prepolymers with different mechanical moduli. Therefore, according to personalized needs, a single polyol or a mixture of several polyols can be used to provide a variety of mechanical modulus products.

[0057] Example 4 In vitro cell experiment of PCL printed samples

[0058] The DLP printed PCL scaffold prepared in Example 3 was sterilized by irradiation and then used. Fresh mesenchymal stem cells were extracted from the bone marrow cavity of 7-day-old SD rats. After culture and passage, the second generation of mesenchymal stem cells were cultured at a concentration of 2×10 6 The density of cells was dropped onto the surface of the scaffold, and finally the scaffold sample seeded with mesenchymal stem cells was placed in a cell culture incubator for 7 days, with the culture medium changed every 2 days. After 7 days, the scaffold sample seeded with mesenchymal stem cells was fixed with 4% paraformaldehyde for 10 minutes, and then washed 3 times with phosphate buffer; then immersed in Triton X-100 membrane permeabilization solution for 10 minutes, and then washed 3 times with PBS; then immersed in Phalloidin-TRITC solution with a concentration of 1 microgram per milliliter at room temperature to stain the cytoskeleton for about 1 hour, and then washed 3 times with PBS; then immersed in DAPI solution with a concentration of 5 micrograms per milliliter at room temperature to stain the cell nucleus for about 10 minutes, and then washed 3 times with PBS; finally, the stained scaffold sample was photographed using a laser confocal device, and the results are shown in the figure below. Figure 6 As shown in A. It can be clearly observed through the fluorescent staining photos that mesenchymal stem cells have a good growth state on the surface of the printed PCL.

[0059] In addition, samples were taken on the 1st, 4th and 7th days of the culture process, and the cell viability was detected by CCK-8 method. CCK-8 reagent was added to the samples and incubated with the cells for 3 hours. The absorbance (OD value) of each sample was measured by microplate reader. The relative viability of each group of cells was calculated based on the absorbance measurement results. Figure 6 As shown in B, mesenchymal stem cells proliferated well on the printed PCL scaffold.

[0060] In general, the double-bond-modified low molecular weight polycaprolactone of this embodiment has good cell compatibility.

Claims

1. A method for preparing double-bond modified low molecular weight polycaprolactone, comprising the following steps: Using a polyol with a functionality n in the range of 2-6 as an initiator and stannous octoate as a catalyst, adjusting the ratio of caprolactone to polyol (the content of caprolactone monomer is m times the hydroxyl equivalent of the polyol, 1≤m≤5) so that the molecular weight of the prepared polycaprolactone precursor is between 300Da and 800Da; After the tube is baked to remove water, evacuated and argon is passed through, potassium carbonate or cesium carbonate is used as an acid-binding agent to introduce an propylene compound into the molecular end of the polycaprolactone precursor to obtain a double-bond-modified low-molecular-weight polycaprolactone reaction product; After the reaction is completed, the reaction product is centrifuged for the first time, filtered, and ethyl acetate is used as an eluent to remove excess potassium carbonate to obtain a clear, transparent, colorless solution. The solvent is then distilled off under reduced pressure, and the product is washed with water thoroughly and centrifuged again to dry the obtained product to obtain a polycaprolactone prepolymer.

2. The method according to claim 1, characterized in that The polyol initiator monomer as the initiator is selected from one or more of ethylene glycol, 1,3-propylene glycol, trimethylolpropane, octanedetol, sorbitol, mannitol and pentaerythritol.

3. The method according to claim 1, characterized in that The first centrifugal acceleration is 500g-1000g, and the time is 10-30 minutes.

4. The method according to claim 1, characterized in that The time for the second centrifugal acceleration of 1000g to 1500g is 20 to 30 minutes.

5. The method according to claim 1, characterized in that The average molecular weight of the obtained polycaprolactone prepolymer is 400-1000Da.

6. The method according to claim 1, characterized in that The obtained polycaprolactone prepolymer has fluidity and a viscosity of 100-300 mpa·s.

7. The method according to claim 1, characterized in that The compression modulus of the polycaprolactone prepolymer sample after photocuring is at the MPa level.

8. Use of the double-bond modified low molecular weight polycaprolactone prepared according to the method of claim 1 in DLP biological 3D printing.

9. A DLP bio-3D printing composition, comprising a double-bonded modified low molecular weight polycaprolactone prepared according to the method of claim 1, a reactive diluent and a photoinitiator.

10. A 3D printed medical device made from the double-bonded modified low molecular weight polycaprolactone prepared according to the method of claim 1.

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