CN114790329A

CN114790329A - High-mechanical-property linear shape memory polyurethane/cellulose nanocrystalline composite material and preparation method and application thereof

Info

Publication number: CN114790329A
Application number: CN202210609108.5A
Authority: CN
Inventors: 罗彦凤; 王远亮; 杨伟
Original assignee: Chongqing University
Current assignee: Chongqing University
Priority date: 2022-05-31
Filing date: 2022-05-31
Publication date: 2022-07-26
Anticipated expiration: 2042-05-31
Also published as: CN114891342A; CN114891342B; CN114790329B

Abstract

The invention provides a high-mechanical-property linear shape memory polyurethane/cellulose nanocrystalline composite material, and a preparation method and application thereof, and belongs to the field of biological materials. The composite material is prepared by taking shape memory polyurethane and cellulose nanocrystals as raw materials, wherein the content of the cellulose nanocrystals is 0.5-20 wt%. The composite material not only maintains excellent shape memory performance, but also greatly improves mechanical property and bone induction effect, and has wide application prospect in preparing bone tissue engineering biological materials.

Description

High-mechanical-property linear shape memory polyurethane/cellulose nanocrystalline composite material and preparation method and application thereof

Technical Field

The invention belongs to the field of biological materials, and particularly relates to a high-mechanical-property linear shape memory polyurethane/cellulose nanocrystalline composite material, and a preparation method and application thereof.

Background

Bone defects caused by severe trauma, tumors, deformities, etc. have been the key and difficult points of clinical orthopedic treatment. In recent years, with the rapid development of tissue engineering, various bone tissue engineering biomaterials have emerged. Among them, linear Shape Memory Polyurethanes (SMPUs) have been considered as a promising biomaterial for bone tissue engineering due to their unique chemical structure and shape memory effect resulting therefrom. The characteristics of alternating soft and hard segments present in SMPUs result in microphase separation, forming nano-to micro-scale islands of hard segment aggregates distributed in continuous soft domains. The phase separation structure can promote osteoblast differentiation and calcification, thereby promoting bone formation. The shape memory effect of SMPUs can be used for minimally invasive implantation and complete filling of bone defects, and bone formation is promoted. However, biodegradable linear shape memory polyurethanes with glass transition temperatures between 37 and 45 ℃ have been reported to have low strength and elastic modulus at room temperature (Chun B., et al., Eur Polymer J.,2006,12, 3367; Marzec M, et al., Materials Science and Engineering: C.2017,80,736; CN 201010239463.5). When the temperature is increased to the physiological temperature (37 ℃) of human bodies, the mechanical property of the polyurethane molecular chain is further reduced due to the enhanced mobility of the polyurethane molecular chain, and the use requirement cannot be met.

The application number 202010081133.1 discloses a biodegradable linear shape memory polyurethane, which has a shape memory temperature (glass transition temperature) of 38-44 ℃ due to the double-ring rigid structure and long-chain hard segment of isosorbide. The linear shape memory polyurethane has a tensile strength of 32.3 to 52.9MPa at 25 ℃ and a tensile strength of 18.1 to 32.3MPa at 37 ℃. The linear shape memory polyurethane has higher mechanical property at room temperature and human physiological temperature, and solves the problems of insufficient mechanical property or overhigh shape recovery temperature of the traditional biodegradable linear shape memory polyurethane at the human physiological temperature to a certain extent. On one hand, however, the mechanical properties of the linear shape memory polyurethane at the physiological temperature (37 ℃) of the human body can not meet the use requirements, and needs to be further improved; on the other hand, the osteoinductive properties of the linear shape memory polyurethane are also to be further improved.

In order to further improve the mechanical properties of the shape memory polyurethane material, the chinese patent application with application number 201910355538.7 discloses a preparation method of a shape memory aqueous polyurethane/cellulose nanocrystalline composite material, comprising: A) heating and dehydrating macromolecular diol, and then stirring and reacting with diisocyanate to obtain a first prepolymer; B) reacting the first prepolymer with a solvent, a micromolecule diol chain extender, a hydrophilic chain extender, an end capping agent and a catalyst to obtain a second prepolymer; C) neutralizing the second prepolymer with a neutralizing agent, reacting with a diamine chain extender in deionized water, dispersing and emulsifying, and removing the solvent to obtain a shape memory aqueous polyurethane emulsion; D) and mixing the cellulose nanocrystal powder dispersion liquid with the shape memory aqueous polyurethane emulsion, and uniformly dispersing by ultrasonic to obtain the shape memory aqueous polyurethane/cellulose nanocrystal composite material. According to the method, the shape memory aqueous polyurethane is prepared, the shape memory aqueous polyurethane and the cellulose nanocrystal are compounded, so that the fixation rate and the recovery rate of the shape memory aqueous polyurethane are improved, and the tensile strength of the material is further improved (552.6-582.6 MPa). However, the mechanical properties of the shape memory aqueous polyurethane/cellulose nanocrystalline composite material still can not meet the requirements of bone repair, because the Young modulus is only 5.0-21.3MPa and is far lower than that of human cortical bone and cancellous bone (3000-30000 MPa and 50-5000 MPa respectively).

In order to promote the application of polyurethane in the field of bone tissue engineering, in particular to the application of load-bearing part repair, it is of great significance to develop a linear shape memory polyurethane material with more excellent mechanical property and osteoinductive property.

Disclosure of Invention

The invention aims to provide a linear shape memory polyurethane/cellulose nanocrystalline composite material with excellent mechanical property and osteoinduction property, and a preparation method and application thereof.

The invention provides a shape memory polyurethane composite material, which is prepared by taking shape memory polyurethane and cellulose nanocrystals as raw materials, wherein the content of the cellulose nanocrystals is 0.5-20 wt%;

the shape memory polyurethane is prepared by taking cyanate coupling agent, macrodiol and catalyst as raw materials to react, wherein the cyanate coupling agent is coupling agent of aliphatic diisocyanate end-capped isosorbide.

Further, the content of the cellulose nanocrystal is 1 wt% to 15 wt%, preferably 10 wt%.

Further, the mole ratio of the cyanate ester coupling agent, the macrodiol and the catalyst is (1.1-2.0): 1: (0.1-0.8), preferably 1.5: 1: 0.5.

further, the preparation method of the cyanate ester coupling agent comprises the following steps: adding aliphatic diisocyanate and isosorbide into a reaction device, adding a solvent for dissolving, adding a catalyst, and reacting in an inert gas environment to obtain the product;

wherein the molar ratio of the aliphatic diisocyanate to the isosorbide is (3-5): 1, preferably 4: 1; the solvent is an organic solvent, preferably DMF; the molar ratio of the isosorbide to the catalyst (400- & gt 600): 1, preferably 500: 1; the reaction temperature is 55-95 ℃, preferably 75 ℃, and the reaction time is 0.5-2h, preferably 1 h; the aliphatic diisocyanate is preferably 1, 6-hexamethylene diisocyanate.

Further, the macrodiol is a product obtained by reacting lactide, polyethylene glycol and a catalyst as raw materials, wherein the molar ratio of the lactide to the polyethylene glycol to the catalyst is (4000-6000): (50-150): 1, preferably 5000:100: 1; the lactide is preferably D, L-lactide, and the polyethylene glycol is preferably PEG 400; the reaction temperature is 120-160 ℃, the reaction time is 140 ℃, and the reaction time is 12-36h, preferably 24 h.

Further, the catalyst is stannous octoate.

Further, the grain diameter of the cellulose nanocrystal is 100nm-35 μm.

The invention also provides a method for preparing the shape memory polyurethane composite material, which comprises the following steps:

(1) uniformly mixing shape memory polyurethane and cellulose nanocrystals to obtain a mixture;

(2) and (3) fusing and mixing the mixture, and molding to obtain the shape memory polyurethane composite material.

Further, in the step (1), the uniform mixing is carried out by mixing for 3-7 minutes by using a grinder;

in the step (2), the temperature of the fusion mixing is 90-130 ℃, and the time is 3-7 minutes; the molding mode is injection molding or hot press molding.

The invention also provides the application of the shape memory polyurethane composite material in preparing bone tissue engineering biological materials.

The CNCs/ISO2-PU composite material is prepared by physically premixing CNCs particles and ISO2-PU powder, then performing melt extrusion in a counter-rotating double-screw extruder, and molding, wherein the CNCs/ISO2-PU composite material has the following beneficial effects:

compared with the ISO2-PU material, the CNCs/ISO2-PU composite material has greatly improved mechanical properties, and particularly has sigma when tested at physiological temperature (37℃) _t And E _t The maximum increase was 36.7% and 89.7%, respectively.

Compared with ISO2-PU, the thermal stability of the CNCs/ISO2-PU composite material is improved.

③ compared with ISO2-PU, T of CNCs/ISO2-PU composite material _g Without obvious change, the composite material of the invention maintains excellent shape memory performance within the ideal temperature range of biomedical shape memory polymer materials.

Compared with PDLLA materials and ISO2-PU materials approved by FDA for bone tissue engineering, the CNCs/ISO2-PU composite material with 10 wt% of CNCs content has obviously enhanced bone induction effect and osteogenic capacity.

The CNCs particles in the invention can be better dispersed in the matrix due to the lower specific surface area when the CNCs particles are fused and compounded with the thermoplastic polymer, and experimental results also show that the CNCs particles in the CNCs/ISO2-PU composite material are very uniformly dispersed in the ISO2-PU matrix, and no hydrogen bond is formed between the CNCs particles and the ISO2-PU long hard segment.

Sixthly, the method for preparing the CNCs/ISO2-PU composite material avoids using a large amount of organic solvent, is more environment-friendly and is easier for industrial production.

In conclusion, the CNCs/ISO2-PU composite material provided by the invention has a wide application prospect in preparation of bone tissue engineering biomaterials.

It will be apparent that various other modifications, substitutions and alterations can be made in the present invention without departing from the basic technical concept of the invention as described above, according to the common technical knowledge and common practice in the field.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 is a scheme showing the synthesis scheme of PDLLA-PEG400-PDLLA macrodiol.

FIG. 2 is a schematic diagram of the preparation of CNCs/ISO2-PU composite material.

FIG. 3 is an infrared spectrum of CNCs, ISO2-PU and CNCs/ISO2-PU composites, wherein (a) and (c) are respectively the corresponding region enlargements in (b).

FIG. 4.110 ℃ storage modulus (G') as a function of strain for ISO2-PU and CNCs/ISO2-PU composites (5 wt% and 15 wt%).

FIG. 5.110 deg.C (a) of ISO2-PU and CNCs/ISO2-PU composites: storage modulus (G') and (b): complex viscosity (η) plotted against shear frequency.

Fig. 6 SEM pictures of CNCs particles.

FIG. 7 SEM (a-e) and optical microscopy pictures (f-j) of CNCs/ISO2-PU composites with CNCs content: 0 wt% (a, e), 1 wt% (b, f), 5 wt% (c, g), 10 wt% (d, h) and 15 wt% (e, j).

FIG. 8 tensile stress strain curves at 25 deg.C (a) and 37 deg.C (b) for ISO2-PU and CNCs/ISO2-PU composites.

FIG. 9 storage modulus (a) and tan delta (b) curves obtained by DMA testing for ISO2-PU and CNCs/ISO2-PU composites (CNCs content 5 wt%, 10 wt% and 15 wt%).

FIG. 10 DSC curve (a), TGA curve (b) and DTG curve (c) for ISO2-PU and CNCs/ISO2-PU composites.

FIG. 11 is a graph of ISO2-PU (a) and CNCs/ISO2-PU composite 2D shape memory process with CNCs content of 5 wt% (b),10 wt% (c) and 15 wt% (D).

FIG. 12, ISO2-PU (a) and CNCs/ISO2-PU composite 3D shape memory process diagram with CNCs content of 5 wt% (b),10 wt% (c) and 15 wt% (D).

FIG. 13 shape memory of CNCs/ISO2-PU composites (CNCs content 10 wt%) at 60 ℃.

FIG. 14 ALP activity after co-cultivation of OBs with PDLLA, ISO2-PU and CNCs/ISO2-PU composites (CNCs content 10 wt%) ((p < 0.05; p < 0.001)).

FIG. 15 ARS staining of calcium deposits (scale 500 μm) after 21 days of co-cultivation of OBs with PDLLA, ISO2-PU and CNCs/ISO2-PU composites (CNCs content 10 wt%).

FIG. 16 SEM pictures of film surface morphology of (a) PDLLA, (b) ISO2-PU and (c) CNCs/ISO2-PU composite (CNCs content 10 wt%) used in vitro mineralization experiments.

Detailed Description

The raw materials and equipment used in the invention are known products, and are obtained by purchasing products sold in the market.

D, L-lactide: the purity is more than or equal to 99 percent; isosorbide (ISO): Sigma-Aldrich, USA, analytically pure; stannous octoate (Sn (Oct) ₂ ) Sigma-Aldrich, USA, analytically pure; 1, 6-Hexamethylene Diisocyanate (HDI): the Aladdin Biotechnology GmbH, analytically pure; n, N-Dimethylformamide (DM)F) The method comprises the following steps The Aladdin Biotechnology Ltd, Anhydrous; cellulose Nanocrystal (CNCs) particles were purchased from Cisco technology nanomaterials Inc. at a Zeta potential of about-50 eV.

Example 1: preparation of CNCs/ISO2-PU composite material

Step 1: preparation of coupling agent HDI-ISO-HDI

HDI and ISO were added to a round bottom single neck flask in a molar ratio of 4:1 while a magnetic stirrer was placed, in m (ISO, g): anhydrous DMF was added into a single-neck bottle at a ratio of 1:6 (DMF, mL), and ISO was completely dissolved by magnetic stirring; further mixing with Sn (Oct) ₂ Adding Sn (Oct) in a molar ratio of 500:1 ₂ After three times of nitrogen replacement, reacting for 1h at 75 ℃ under the protection of nitrogen; and after the reaction is finished, cooling to room temperature, and removing unreacted HDI by using normal hexane dried by a molecular sieve to obtain white powder, drying to constant weight, namely the HDI-blocked ISO novel diisocyanate coupling agent which is named as HDI-ISO-HDI.

And 2, step: preparation of macrodiol PDLLA-PEG400-PDLLA

Mixing D, L-lactide, PEG400, Sn (Oct) ₂ Adding the mixture into a round bottom single-mouth bottle with a magnetic stirrer according to the mol ratio of 5000:100:1, vacuumizing for 30min, and sealing; putting the single-mouth bottle into an oil bath pan at 140 ℃, starting magnetic stirring after the mixture is completely melted to uniformly stir the reaction system, and continuously reacting for 24 hours; after the reaction is finished, a dichloromethane/ice absolute ethyl alcohol (-15 ℃, the volume ratio of dichloromethane to ice absolute ethyl alcohol is 1: 10) coprecipitation system is adopted for repeatedly purifying the product for three times, and then the dichloromethane/ice normal hexane (-15 ℃, the volume ratio of dichloromethane to ice normal hexane is 1: 8) coprecipitation system is used for purifying once; vacuum drying at room temperature for 72h to obtain target macrodiol, named PDLLA-PEG 400-PDLLA. The synthetic route of PDLLA-PEG400-PDLLA is schematically shown in FIG. 1.

And step 3: preparation of polyurethane Material ISO2-PU

HDI-ISO-HDI and PDLLA-PEG400-PDLLA were added to a round bottom four-neck flask with a stir bar at a molar ratio of 1.5:1.0 while mixing with m (PDLLA-PEG400-PDLLA, g): adding anhydrous DMF at a ratio of 5:4 (DMF, mL) into the solution, and stirring the solution to completely dissolve the solid; then PDLLA-PEG400-PDLLA and Sn (Oct) ₂ Adding Sn (Oct) at a molar ratio of 500:1 ₂ Reacting for 3 hours at 75 ℃ under the protection of nitrogen; finally adding ISO in the molar ratio of PDLLA-PEG400-PDLLA to ISO of 1.0:0.5, and continuing to react for 16 h; after the reaction is finished, cooling to room temperature, diluting with dichloromethane, precipitating and purifying the product in absolute ethyl alcohol twice (the volume ratio of the diluted solution to the absolute ethyl alcohol is 1: 10), and purifying the product once by using a dichloromethane/n-hexane coprecipitation system (the volume ratio of dichloromethane to n-hexane is 1: 8). And (3) drying the purified product in vacuum to constant weight to obtain the polyurethane material named as ISO-PUs.

And 4, step 4: CNCs/ISO2-PU composite material prepared by injection molding process

The powdered CNCs granules were dried under vacuum at 60 ℃ for 2 days and ISO2-PU powder was dried under vacuum at 40 ℃ for 5 days. Then, different proportions of ISO2-PU powder were mixed with the CNCs for 5min using a grinder at a speed of 1800rpm to give a mixture in which the content of CNCs in the final mixture was controlled to be 1 wt%, 3 wt%, 5 wt%, 7 wt%, 10 wt%, 15 wt%, respectively. Next, the mixture was melt-mixed in a counter-rotating twin-screw extruder, and the mixing was carried out at a temperature of 110 ℃ for 5min at a speed of 60 rpm. Finally, the CNCs/ISO2-PU composite material is obtained by injection molding processing of an injection molding machine, and the injection molding conditions are as follows: keeping the temperature at 120 ℃ for 30s, and simultaneously keeping the pressure at 10MPa and the mold temperature at 30 ℃.

Example 2: preparation of CNCs/ISO2-PU composite material

Step 1-3: the same as in example 1.

And 4, step 4: preparation of CNCs/ISO2-PU composite material by hot-press molding process

The powdered CNCs particles were dried under vacuum at 60 ℃ for 2 days and ISO2-PU powder was dried under vacuum at 40 ℃ for 5 days. Then, ISO2-PU powder was mixed with CNCs in various proportions at a speed of 1800rpm for 5min using a grinder to give a mixture in which the content of CNCs in the final mixture was controlled to be 1 wt%, 3 wt%, 5 wt%, 7 wt%, 10 wt%, 15 wt%, respectively. Next, the mixture was melt-mixed in a counter-rotating twin-screw extruder, and the mixing was carried out at a temperature of 110 ℃ for 5min at a speed of 60 rpm. Finally, the CNCs/ISO2-PU composite material is hot-pressed into a film at 120 ℃ for 5min by a hot plate.

The beneficial effects of the present invention are demonstrated by the following experimental examples.

Experimental example 1: infrared Spectrum testing

1. Test sample

Pure CNCs, ISO2-PU and CNCs/ISO2-PU composite material prepared by hot press molding.

2. Experimental methods

Fourier transform infrared spectrometer (Nicolet iS50, Thermo Fisher Scientific) was used to record the sample size at 400-4000 cm ^-1 FT-IR (transmission) spectrum of range with resolution of 4cm ^-1 Each sample was scanned 16 times.

3. Results of the experiment

FIG. 3 is the FT-IR spectra of pure CNCs, ISO2-PU and CNCs/ISO2-PU composites. In pure CNCs, 3250-3500 cm ^-1 The strong absorption peak of (2) is the stretching vibration peak of-OH group on the surface of CNCs, and the length of the absorption peak is 2897cm ^-1 The absorption peak at (A) is-CH in CNCs ₂ Stretching vibration peak of radical, 1647cm ^-1 The absorption peak at (a) is the absorption peak of adsorbed water in the CNCs. For the spectrum of ISO2-PU, 1754cm ^-1 The absorption peak belongs to the stretching vibration peak of an ester-C ═ O group, and is 2800-3040 cm ^-1 The absorption peak at (A) is-CH in PDLLA-PEG400-PDLLA triblock macrodiol (soft segment of ISO 2-PU) ₂ -and-CH ₃ Stretching vibration peak of radical. At the same time, 3300- ^-1 、1600-1731cm ^-1 And 1526cm ^-1 The peaks at (a) belong to characteristic absorption peaks of the-NH group, the amide I band (-C ═ O group), and the amide II band (-NH group) in the urethane bond, respectively. Wherein, 1731cm ^-1 The absorption peak at (A) is a characteristic absorption peak of free-C ═ O in a urethane bond, and 1630cm ^-1 And 1650cm ^-1 Peaks at (A) are ordered and disordered hydrogen bond carbamates-C ═ O, respectively, which are at 1754cm ^-1 The peak of stretching vibration of the ester-C ═ O group at (b) partially overlaps. 1630-1650cm after addition of CNCs particles in ISO2-PU ^-1 The intensity of the peak at (a) gradually decreases as the content increases, and almost disappears after the content of CNCs exceeds 5 wt%. At the same time, 3320cm in the spectrum of pure ISO2-PU ^-1 (formation of ordered Hydrogen bonds-NH) and 3340cm ^-1 The absorption peak at (disordered hydrogen bond-NH) is obviously higher than 3400cm ^-1 Absorption peak at (free-NH). Addition of CNCs to 3320cm ^-1 The peak intensity gradually decreases and almost disappears after the content of CNCs exceeds 5 wt%. Furthermore, with further increase in the content of CNCs, 3340cm ^-1 Gradually higher than 3400cm ^-1 A peak at (c), which may be due to a large increase in-OH groups in the composite.

The results show that the CNCs/ISO2-PU composite material added with the CNCs is successfully synthesized by the method.

Experimental example 2: rheological Property test

1. Test sample

CNCs, ISO2-PU and CNCs/ISO2-PU composite material prepared by hot press molding.

2. Experimental method

The rheological measurements were performed on an AR-G2 rotational rheometer (TA Instruments, USA) equipped with parallel plate diameters of 25 mm. The composite material for rheological measurement was processed into a sheet material having a diameter of 25mm and a thickness of 1mm by injection molding. The sample was melted at the measurement temperature for 3 minutes before the start of the measurement.

3. Results of the experiment

The rheological test temperature of ISO2-PU and CNCs/ISO2-PU composites (CNCs content 5 wt% and 10 wt%) was set at 110 ℃ and the dynamic strain sweep curve obtained is shown in FIG. 4. It can be seen that the strain curves for the three materials are linear from 0% to 100%, so that the subsequent dynamic frequency sweep test selects a 10% strain, i.e. at 110 ℃ and 10% strain.

The storage modulus (G') and complex viscosity (. eta.) of the resulting ISO2-PU and CNCs/ISO2-PU composite melts from the dynamic frequency sweep test are shown in FIG. 5 as a function of angular frequency. In fig. 5(a), the G' curve as a whole gradually becomes a straight line as the CNCs content increases, and the slope gradually decreases. In the low frequency region, addition of 1 wt% CNCs also significantly enhanced G ', and G' increased slowly with further addition of CNCs (1 wt% to 5 wt%). However, when the CNCs content increases from 5 wt% to 7 wt%, G' suddenly increases sharply, and the increase becomes slow again after the content exceeds 7 wt% until the CNCs content reaches 15 wt%. In fig. 5(b), η of pure ISO2-PU exhibits newtonian fluid behavior at lower frequencies characterized by η being independent of shear rate (response with change in shear rate is unchanged), and at higher frequencies, significant shear thinning behavior. The addition of the CNCs particles gradually reduces the Newtonian plateau, while a stronger shear-thinning behavior in the high frequency region than pure ISO2-PU can be observed for all CNCs/ISO2-PU composites. When the content of CNCs reached 7 wt%, the plateau completely disappeared and the curve tended to be a straight line. Furthermore, η of the composite material in the low frequency region increases with the increase of the content of CNCs as with G', but increases relatively slowly until the content of CNCs reaches 7 wt% at a content of 1 to 5 wt%.

Experimental example 3: characterization of microscopic morphology

1. Test sample

CNCs, ISO2-PU, injection-molded CNCs/ISO2-PU composite material sample bars (in the shape of standard dog bones, the effective size of 20.0X 4.0X 2.0mm (according to ISO 527-2-5A), and subjected to annealing at 50 ℃ for 20min) and CNCs/ISO2-PU composite material sheets (the thickness is about 20 μm) prepared by hot press molding.

2. Experimental methods

And (4) SEM test: the cross-sectional morphology of the composite samples (bars for mechanical testing) was observed by a Quattro S Scanning Electron Microscope (SEM) (ThermoFisher Scientific, USA) at a voltage of 8 kV. The cross section of the sample was brittle with liquid nitrogen and plated with gold before testing.

And (3) testing by an optical microscope: dispersion of spherical CNCs agglomerate particles in ISO2-PU matrix sheets of CNCs/ISO2-PU composites were also observed and recorded using an optical microscope (mingmei photoelectric) equipped with a camera.

3. Results of the experiment

In order to observe the dispersion of the CNCs particles in the ISO2-PU matrix, the morphology of the liquid nitrogen brittle fracture cross section of the CNCs/ISO2-PU thin film was observed using SEM. The SEM appearance of the CNCs particles is shown in FIG. 6, and the particles are spherical and have diameters ranging from 100nm to 35 μm. The profile of the pure ISO2-PU and CNCs/ISO2-PU composites is shown in FIG. 7. ISO2-PU (FIG. 7(a)) exhibited a smooth cross-section, whereas the composite material with a 1 wt% CNCs content (FIG. 7(b)) had a rough cross-section, but no visible agglomeration of CNCs particles occurred. When the CNCs content was further increased to 5 wt% (fig. 7(c)) the cross-section became coarser and the CNCs particles were tightly packed in the ISO2-PU matrix. When the CNCs content reached 10 wt% (fig. 7(d)), more spherical particles were observed and significant cracks appeared in the cross-section, indicating that the obtained CNCs/ISO2-PU composite material became significantly brittle. For composites with 15 wt% CNCs content (fig. 7(e)), some significant aggregation of matrix fragments and CNCs particles was observed at the brittle fracture surface, the presence of fragments indicating a further increase in brittleness of the composite.

In addition, the CNCs particles have high crystallinity, while ISO2-PU is amorphous, and the light transmittance of the CNCs particles is obviously different from that of the ISO2-PU, so that the CNCs particles can be observed in a dispersion state under an optical microscope. The pure ISO2-PU exhibits typical phase separation morphology between soft and hard segments (FIG. 7(f)), where the hard segments form rounded aggregates of size 2-8 μm and are uniformly dispersed in the soft segment phase. The addition of 1 wt% of particles of CNCs severely disrupts the phase separation in ISO2-PU and no hard segment aggregates are observed in the composite (FIG. 7(g)), but a homogeneous dispersion of individual spherical particles of CNCs in the matrix of ISO2-PU can be observed. Little spherical particle aggregation was observed when the CNCs content was 5 wt% (fig. 7 (h)). As the CNCs content increased to 10 wt% (fig. 7(i)), more CNCs particles aggregated could be observed, but the number of particles aggregated was still much less than the single dispersed particles. When the content of CNCs increased to 15 wt% (FIG. 7(j)), significant aggregation of CNCs particles occurred.

The above experimental results show that when the addition amount of CNCs is less than 15 wt%, the CNCs particles in the CNCs/ISO2-PU composite material are dispersed in the ISO2-PU matrix very uniformly.

Experimental example 4: mechanical and thermomechanical property characterization

1. Test sample

ISO2-PU and CNCs/ISO2-PU composites. Mechanical test specimens were injection molded in the shape of standard dog bones with effective dimensions of 20.0X 4.0X 2.0mm (according to ISO 527-2-5A). Thermo-mechanical performance (DMA) test specimens were hot-pressed films with dimensions of 0.1 × 5.0 × 50.0 mm.

2. Experimental methods

The mechanical properties of the composite material are performed on a UTM5305SYXL type electronic universal material testing machine (Shenzhen Sansi) which is provided with a heating cavity plate and an electronic extensometer. The loading rates for the tests were all 5.0mm/min, the test temperatures were 25 ℃ and 37 ℃, and the final result was the average of 5 replicates. The DMA testing of the composites was performed in tensile mode on a DMA-Q800(TA Instruments, USA) dynamic mechanical Analyzer. The test conditions were constant frequency: 1 Hz; pre-stressing: 0.01N; strain: 0.1 percent; test temperature range: -10 to 90 ℃; the heating rate is as follows: 2 ℃/min.

3. Results of the experiment

TABLE 1 mechanical properties of ISO2-PU and CNCs/ISO2-PU composites at 25 ℃ and 37 ℃

*σ _t The tensile strength; e _t Young's modulus; epsilon _t Elongation at break.

The tensile stress-strain curves at 25 ℃ and 37 ℃ for ISO2-PU and CNCs/ISO2-PU composites are shown in FIG. 8, while the mechanical properties calculated from the corresponding stress-strain curves are listed in Table 1. The addition of particles of CNCs significantly improved the mechanical properties of ISO2-PU when tested at 25 ℃ (fig. 8(a)), and this trend of enhancement continued with the increase in CNCs content. Young's modulus (E) of pure ISO2-PU _t ) And tensile strength (. sigma.) _t ) 3498.3MPa and 50.1MPa respectively, and E when the content of CNCs in the CNCs/ISO2-PU composite material is 15 wt% _t And σ _t Increasing to 4569.2MPa and 62.5MPa respectively. Simultaneous elongation at break (. epsilon.) _t ) From 6.8% of pure ISO2-PU, it gradually decreased to 3.4% (containing 15 wt% CNCs). Furthermore, all samples were at 25 deg.CThe tensile stress-strain curves of (a) all show significant yield, but no complete necking phase and plateau region occurs.

In contrast to the tensile stress-strain curve at 25 ℃ in FIG. 8(a), the curve at 37 ℃ (human physiological temperature) in FIG. 8(b) shows typical ductile polymer characteristics, i.e., a distinct yield point, long necking phases in the curve, and a long plateau for both the composites, except for composites with 10 wt% and 15 wt% CNCs content, and for pure ISO 2-PU. Epsilon of all samples _t The values are all significantly higher than at 25 ℃ (Table 1), and ISO 2-composite epsilon with 1 wt% to 5 wt% content of PU and CNCs _t The values differ a little, but when the content of CNC exceeds the percolation threshold (between 5% and 7% by weight), ε _t The value drops sharply from-190% to-25%. E at 37 ℃ compared with 25 ℃ _t And σ _t Are significantly reduced. However, E of CNCs/ISO2-PU composites _t And σ _t Still much higher than pure ISO2-PU, the composite material with 15 wt% CNCs content _t Is-40.6 MPa, E _t 2021.1 MPa.

Based on the above mechanical properties, the thermodynamics of pure ISO2-PU and CNCs/ISO2-PU composites (CNCs content 5 wt%, 10 wt% and 15 wt%) were further investigated using DMA. FIG. 9(a) is a graph showing the change in storage modulus (E') with temperature. It is clear that the addition of particles of CNCs in the glassy state significantly increases the E' of ISO2-PU, and that the higher the content of CNCs in the composite, the higher the entry into T _g The higher the temperature in the zone. Meanwhile, at 25 ℃ (glassy state region), E' of the composite material with 15 wt% of CNCs content is significantly higher than that of the composite material with 10 wt% of CNCs content; however, when at 37 ℃ (T) _g Zone), the E 'curves of the two composites almost completely coincide, and they differ more from the E' of pure ISO2-PU than 25 ℃, which is consistent with the results of tensile mechanical property tests. In addition, composites with higher CNCs content are at T _g The faster zone E' falls, so that the T of the pure ISO2-PU and CNCs/ISO2-PU composites _g Zones all end at 60 ℃. Therefore, the temperature (T) of the subsequent shape memory test _tran ) Optionally 60 deg.C ^[31] Shape fixation temperature (T) _fix ) The temperature was set to 20 ℃. FIG. 9(b) For the tan delta curve with temperature, only one tan delta peak is observed for each sample, and the T is obtained from the peak _g The values do not change significantly with the addition and increase of the content of CNCs, the T of pure ISO2-PU and CNCs/ISO2-PU composites _g The values are all between 54 ℃ and 57 ℃. However, the intensity of the tan δ peak decreases with increasing CNCs content.

The experimental results show that compared with the ISO2-PU material, the CNCs/ISO2-PU composite material has greatly improved mechanical properties, and particularly has sigma when tested at physiological temperature (37℃) _t And E _t The maximum increase was 36.7% and 89.7%, respectively. In the CNCs/ISO2-PU composite material, the composite material with the content of 10 wt% and 15 wt% of CNCs has better mechanical property.

Experimental example 5: characterization of thermal Properties

1. Test sample

ISO2-PU and CNCs/ISO2-PU composites.

2. Experimental method

DSC measurement of N on DSC-Q100(TA Instruments, USA) ₂ Is carried out in an atmosphere. Firstly, heating a sample from room temperature to 150 ℃ at a heating rate of 20 ℃/min, carrying out isothermal treatment for 3min, and then cooling to-20 ℃ at a cooling rate of-20 ℃/min. Finally, the sample was heated again to 150 ℃ at a heating rate of 10 ℃/min. Glass transition temperature (T) _g ) Determined by the second heating scan profile. Thermogravimetric analysis (TGA) was performed on a TGA-Q50(TA Instruments, USA) with samples on N ₂ The temperature is raised from 25 ℃ to 600 ℃ in the atmosphere at a heating rate of 10 ℃/min.

3. Results of the experiment

FIG. 10(a) is a DSC curve of pure ISO2-PU and CNCs/ISO2-PU composites, from which T is obtained _g Value and Heat Capacity variation (. DELTA.C) _p ) The values are shown in Table 2.ISO 2-T of PU _g Value of 42.2 ℃ T of the composite material after addition of CNCs particles _g The value is slightly increased but is not significant, the content of CNCs is increased from 1 wt% to 15 wt% of the composite material T _g The values only fluctuate between 43.0 ℃ and 43.7 ℃. At the same time, Δ C of ISO2-PU _p 0.501J/(g.K), Δ C of the composite with increasing CNCs content _p Is gradually reducedΔ C of the composite when the CNCs content is 15% _p The temperature is reduced to 0.398J/(g.K).

TABLE 2 thermal Properties of ISO2-PU and CNCs/ISO2-PU composites

T _g : a glass transition temperature; delta C _p : hot melting change; t is a unit of ₀ : an initial decomposition temperature; t is a unit of _fastest : the fastest decomposition temperature.

The thermal stability of ISO2-PU and CNCs/ISO2-PU composites was evaluated by TGA testing, and the TGA and DTG curves obtained are shown in FIGS. 10(b) and (c). There are two weight loss stages in each curve of the CNCs/ISO2-PU composite, whereas ISO2-PU has only one weight loss stage. The first weight loss stage of the CNCs/ISO2-PU composite material occurs at the temperature of 100-150 ℃, and the second weight loss stage is highly coincident with the weight loss stage of ISO2-PU and both occur at the temperature of 260-420 ℃. Corresponding initial decomposition temperatures (T) obtained from TGA and DTG curves ₀ 5% weight loss) and the fastest decomposition temperature (T) _fastest ) As shown in table 2.

The above experimental results show that the T of CNCs/ISO2-PU composite material is compared with that of ISO2-PU _g No significant change, still within the ideal temperature range of the biomedical shape memory polymeric material; meanwhile, the thermal stability of the CNCs/ISO2-PU composite material is improved.

Experimental example 6: characterization of shape memory Properties

1. Test sample

ISO2-PU and CNCs/ISO2-PU composites.

2. Experimental methods

The shape memory test was performed under strain control in tensile mode on DMA with a sample size of 0.1 × 5.0 × 50.0 mm. The specific procedure is as follows: increasing the temperature from 25 ℃ to the shape memory temperature (T) at a temperature rise rate of 5 ℃/min under the condition that the loading strain is 0.1 percent _tran ) (ii) a (II) after 5min of isothermal heating, increasing the strain to 50% at a rate of 5%/min and keeping for 5 min; (III)Cooling to below T at a cooling rate of 5 ℃/min _tran Shape fixing temperature (T) of 30 DEG C _fix ) Keeping for 5 min; (IV) removing the stress and raising the temperature to T again at a temperature raising rate of 5 ℃/min _tran And keeping the temperature constant for 40min to finish the recovery process. Shape memory Properties of samples in terms of shape fixation Rate (R) _f ) And shape recovery ratio (R) _r ) And (3) evaluating, wherein the calculation formulas are shown as a formula (1) and a formula (2).

Wherein epsilon ₁ For strain imposed on the sample,. epsilon ₂ To cool to T _fix And strain, epsilon, after removal of stress ₃ The strain after recovery is isothermal for 40 min.

3. Results of the experiment

According to the DMA test result, the shape memory temperature (T) _tran ) Set at 60 ℃ and a shape fixing temperature (T) _fix ) Set at 20 ℃. The 2D and 3D form-fix-recovery cycle plots for pure ISO2-PU and CNCs/ISO2-PU composites (CNCs content of 5 wt%, 10 wt% and 15 wt%) are shown in fig. 11 and fig. 12, respectively. ISO 2-fixed Rate (R) of PU _f ) 99.8% addition of CNCs to R of the composite _f Without significant influence, 5 wt%, 10 wt% and 15 wt% of ε of CNCs/ISO2-PU composites ₂ 49.8%, 49.9% and 49.9%, respectively, i.e. their R _f The values were 99.6%, 99.8% and 99.8% in this order. However, the recovery (R) of CNCs/ISO2-PU composites _r ) The effect of CNCs particles was evident. ε of composite containing 5 wt%, 10 wt% and 15 wt% CNCs ₃ 11.0%, 13.6% and 15.1%, respectively, i.e. their R _r The values are, in this order, 78.0%, 72.8% and 69.8%, significantly lower than the R of pure ISO2-PU _r Value (91.2%). At the same time, it is clear that for composites with higher CNCs content, the stress required to achieve a strain of 50.0% is achievedEven lower, this is more evident in the 2D form-fix-recovery cycle plot shown in fig. 11.

The shape memory process of the CNCs/ISO2-PU composite (CNCs content 10 wt%) film is demonstrated in FIG. 13. The sample film was coiled into a spiral shape (temporary shape) after heating at 60 ℃ for 3 min; then cooled at 20 ℃ to fix the shape; then placing the deformed sample in an environment of 60 ℃ again; and finally return almost completely to the original shape within 30 s.

From the above results, it can be seen that when the content of CNCs in the CNCs/ISO2-PU composite material is 10 wt% or less, the particles are uniformly dispersed, but when the content of CNCs is increased to 15 wt%, significant particle agglomeration occurs; the mechanical properties of the composite material with the content of CNCs of 10 wt% and the composite material with the content of CNCs of 15 wt% are similar to and obviously higher than those of other composite materials; the shape memory performance of the composite material with the content of CNCs of 10wt percent is obviously superior to that of the composite material with the content of CNCs of 15wt percent; the composite material having a CNCs content of 10 wt% had a CNCs content of less than that of the composite material having a CNCs content of 15 wt%. Therefore, the comprehensive performance of the CNCs/ISO2-PU composite material with the content of 10 wt% of CNCs is optimal by comprehensively considering the addition amount, the dispersibility, the mechanical property and the shape memory property of the CNCs. The composite material of the component is selected for further in vitro mineralization capability test.

Experimental example 7: characterization of mineralization Performance in vitro

1. Test sample

ISO2-PU and CNCs/ISO2-PU composite material

2. Experimental method

The in vitro mineralization ability of the samples was assessed by alkaline phosphatase (ALP) activity and alizarin red s (ars) staining. Briefly, the material was dissolved in chloroform to make a 30 wt% solution, which was then cast as a film on a 15mm diameter glass plate and dried under vacuum for 7 days under 40 mm glass plates to completely remove the chloroform before use. The samples were UV-irradiated on both sides for 30min, respectively, placed in 24-well plates (material membrane side up), co-cultured in 1 plate (material/cm 2 cell density was inoculated to skull Osteoblasts (OBs) of newborn SD rat suckling mice, co-cultured in high-sugar medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, after 4, 7, 14 days of culture, the medium was aspirated from the 24-well plate, washed 3 times with PBS, then the sample was centrum was moved to clean new wells, 250 was used for each sample, a Triton X-100 lysis buffer at a concentration of 0.2 vol% was extracted from the sample, finally the lysate was centrifuged at 10000r/min for 10min at 4, and the supernatant was collected about 200 clear liquid, ALP activity was calculated by ALP assay kit and absorbance at 562nm of the supernatant was measured using a microplate reader (Bio-Rad 550, California, USA), after 21 days of culture, the medium was aspirated from the 24-well plate and fixed with 4.0% paraformaldehyde in 4 plates for 8 hours. Then washed 3 times with purified water and stained with ARS for 10 minutes at room temperature. Finally, the sample is washed until pure water is colorless. The morphology of the samples was observed and recorded by optical microscopy (Mingmei photoelectricity). All ALP activity and ARS staining experiments were performed in triplicate and the medium was changed every two days during the culture.

3. Results of the experiment

The in vitro mineralization ability of neonatal rat Osteoblasts (OBs) cultured on PDLLA, ISO2-PU and CNCs/ISO2-PU composites (10 wt%) was also assessed by measurement of ALP activity and ARS staining with ISO2-PU and FDA approved PDLLA for bone tissue engineering as controls. ALP activity after co-culturing of OBs and materials for 4, 7 and 14 days is shown in FIG. 14. Overall, ALP activity of OBs on all three materials continuously increases with the culture time, ALP activity of OBs on ISO2-PU is higher than PDLLA, and addition of CNCs particles can further significantly improve ALP activity of OBs. In particular, there was no significant difference in ALP activity of OBs on PDLLA and ISO2-PU after 4 days of co-culture, but the ALP activity of OBs on CNCs/ISO2-PU composite (10 wt%) was already significantly higher than those of the two groups (p > 0.05); after 7 days of co-cultivation, the ALP activity of OBs on ISO2-PU was significantly higher than that of PDLLA group (P >0.05), while the ALP activity of OBs on CNCs/ISO2-PU composite (10 wt%) was further significantly higher than that of PDLLA and ISO2-PU group (P > 0.01); ALP activity was further increased in all three groups after 14 days of co-culture, and the difference in ALP activity between the groups was consistent with that at day 7.

The ARS staining after 21 days co-incubation of OBs on 3 sets of sample films and the corresponding optical images are shown in fig. 15, all images taken at the same light intensity. The shade of the color of the surface of the ARS-dyed samples represents the amount of calcium deposits, red deposits were observed on the film surface of PDLLA, ISO2-PU and CNCs/ISO2-PU composites (10 wt%), but the ISO2-PU film was visually darker than the PDLLA film surface, and the staining of the CNCs/ISO2-PU composite surface was significantly more intense than in the two groups, indicating that the CNCs/ISO2-PU composites (10 wt%) had significantly more calcium deposits on the film surface.

In the in-vitro mineralization experiment, three materials are dissolved by trichloromethane, then are subjected to film casting, are completely removed of the solvent and then are used, and a hot-pressing sample or an injection-molding sample is not directly used for the experiment, so that the influence of grains on OBs caused by a mold on the surface of the hot-pressing sample or the injection-molding sample is avoided. Meanwhile, in order to avoid the enrichment of CNCs particles on the surface of a CNCs/ISO2-PU composite material (CNCs10 wt%) film due to the fact that the density of CNCs is far smaller than that of chloroform, the three materials are prepared into a high-concentration solution with the concentration of 30 wt%, and the CNCs particles are stabilized by using the high viscosity of the composite material solution. The surface topography of the three-material film is shown in fig. 16, the surface of the PDLLA and ISO2-PU film is totally very flat except a few microcracks caused by solvent volatilization, and the CNCs/ISO2-PU composite material (CNCs10 wt%) can be seen on the surface of the film, and the CNCs are uniformly distributed and are not agglomerated or enriched. Thus, the behavior of OBs cells on the surface of such a sample may well be representative of the properties of the material.

Materials with more polar and hydrophilic groups on the surface are generally thought to better promote cell attachment, diffusion and proliferation. Furthermore, the biomaterial surface should generally have a certain roughness to promote cell attachment, except in rare cases (including joints and some soft tissue implants). For osteoblasts, surface roughness is not only an important factor in initiating adhesion, but also an important factor affecting their cellular activity and mineralization. It can be seen from fig. 16 that the addition of the CNCs particles significantly increases the surface roughness of the material, while the CNCs surface also has abundant-OH, which is beneficial to the adhesion, proliferation and mineralization of OBs.

The above experimental results show that, compared with PDLLA material approved by FDA for bone tissue engineering and ISO2-PU material, the CNCs/ISO2-PU composite material with 10 wt% of CNCs content has obviously enhanced osteoinductive effect and osteogenic capacity.

In conclusion, the invention provides a high-mechanical-property linear shape memory polyurethane/cellulose nanocrystalline composite material, and a preparation method and application thereof. The composite material is prepared by taking shape memory polyurethane and cellulose nanocrystal as raw materials, wherein the content of the cellulose nanocrystal is 0.5-20 wt%. The composite material not only maintains excellent shape memory performance, but also greatly improves mechanical property and bone induction effect, and has wide application prospect in preparing bone tissue engineering biological materials.

Claims

1. A shape memory polyurethane composite material is characterized in that: the composite material is prepared by taking shape memory polyurethane and cellulose nanocrystals as raw materials, wherein the content of the cellulose nanocrystals is 0.5-20 wt%;

2. The shape memory polyurethane composite of claim 1, wherein: the content of the cellulose nanocrystal is 1 wt% -15 wt%, and preferably 10 wt%.

3. The shape memory polyurethane composite of claim 1, wherein: the mole ratio of the cyanate coupling agent to the macrodiol to the catalyst is (1.1-2.0): 1: (0.1-0.8), preferably 1.5: 1: 0.5.

4. the shape memory polyurethane composite of claim 1, wherein: the preparation method of the cyanate ester coupling agent comprises the following steps: adding aliphatic diisocyanate and isosorbide into a reaction device, adding a solvent for dissolving, adding a catalyst, and reacting in an inert gas environment to obtain the product;

5. The shape memory polyurethane composite of claim 1, wherein: the macrodiol is a product obtained by reacting lactide, polyethylene glycol and a catalyst as raw materials, wherein the molar ratio of the lactide to the polyethylene glycol to the catalyst is (4000) -6000): (50-150): 1, preferably 5000:100: 1; the lactide is preferably D, L-lactide, and the polyethylene glycol is preferably PEG 400; the reaction temperature is 120-160 ℃, the reaction time is 140 ℃, and the reaction time is 12-36h, preferably 24 h.

6. Shape memory polyurethane composite material according to any of claims 1 to 5, characterized in that: the catalyst is stannous octoate.

7. Shape memory polyurethane composite material according to any of claims 1 to 5, characterized in that: the grain diameter of the cellulose nanocrystal is 100nm-35 mu m.

8. A process for preparing a shape memory polyurethane composite according to any one of claims 1 to 7, characterized in that: the method comprises the following steps:

(2) and fusing, mixing and molding the mixture to obtain the shape memory polyurethane composite material.

9. The method of claim 8, wherein: in the step (1), the uniform mixing mode is to mix for 3-7 minutes by a grinder;

in the step (2), the temperature of the fusion mixing is 90-130 ℃, and the time is 3-7 minutes; the forming mode is injection molding or hot press molding.

10. Use of the shape memory polyurethane composite of any one of claims 1 to 7 for the preparation of a bone tissue engineering biomaterial.