CN114656678A

CN114656678A - Use of porous polymer film with regular pores for preparing artificial periosteum

Info

Publication number: CN114656678A
Application number: CN202210333364.6A
Authority: CN
Inventors: 罗彦凤; 杨伟; 王远亮
Original assignee: Chongqing University
Current assignee: Chongqing University
Priority date: 2022-03-31
Filing date: 2022-03-31
Publication date: 2022-06-24
Anticipated expiration: 2042-03-31
Also published as: CN114656678B

Abstract

The invention belongs to the technical field of polymer materials, and particularly relates to application of a porous polymer film with regular pores in preparation of an artificial periosteum. The shape memory polyurethane provided by the invention is prepared from diisocyanate, soft block polymer and

polymerized; the soft segment polymer is polylactic acid, polyglycolic acid, polycaprolactone, polylol or a copolymer of at least two of the polylactic acid, the polyglycolic acid, the polycaprolactone and the polylol. Combining this shape memory polyurethane material with a water drop templating method enables the formation of porous polymer films containing crystalline structures. The porous film material has excellent mechanical properties, can meet the performance requirements of application scenes such as artificial periosteum and the like, and has good application prospects.

Description

Use of porous polymer film with regular pores for preparing artificial periosteum

Technical Field

The invention belongs to the technical field of polymer materials, and particularly relates to application of a porous polymer film with regular pores in preparation of an artificial periosteum.

Background

Shape Memory Polymer (SMP), also called Shape Memory Polymer, refers to a Polymer material that can restore its original Shape after a product with the original Shape is fixed after its original condition is changed under a certain condition and stimulated by external conditions (such as heat, electricity, light, chemical induction, etc.). Shape memory polymers have been widely used in biomedical, aerospace, optical and textile fields due to their shape recovery properties.

Shape memory polymers include thermotropic, electroluminescent, photoinduced, chemosensory, etc. according to their recovery principle. The thermotropic shape memory polymer can be adjusted to be consistent with the body temperature by controlling the glass transition temperature of the thermotropic shape memory polymer, thereby realizing the application in the medicine. For example: the membrane made of the thermotropic shape memory polymer material or the device with a specific shape can be miniaturized and deformed, and then is implanted into the body through a micro-catheter, and the original set shape is recovered after the membrane reaches the correct position.

Thermotropic shape memory polymers include the polyurethane, ethylene/vinyl acetate copolymers, and crosslinked polyethylene, among many classes. When the materials are used for preparing films or devices implanted into human bodies, a series of performance requirements such as permeability, biocompatibility, mechanical property and the like need to be met according to the implanted positions and purposes. The conventional thermotropic shape memory polymer material made into a common compact membrane can not meet the requirements of permeability and biocompatibility. To ameliorate this problem, porous polymer films with regular pores have the potential to be membranes for implantation into the human body due to their unique performance characteristics (e.g., permeability to molecules/ions, biocompatibility, extensibility, etc.).

However, the existing thermotropic shape memory polymer material can cause the problem of insufficient mechanical property in the process of introducing a large number of pores to prepare a porous membrane. For example, "CN 201110224930.1 a method for forming a polyurethane porous film" provides a method for preparing a polyurethane porous film by using a water droplet template method. However, due to the limitation of mechanical properties, the porous polymer films made of these polyurethane materials are still difficult to be applied in the fields of bionics, tissue engineering, etc. with high requirements on the mechanical properties of the materials. The method has great limitation on the development and application of the porous polymer film, and the design and preparation of the polymer film with good mechanical property and a regular porous structure are beneficial to expanding the application of the material. The ordered arrangement, crystal or microphase separation of polymer molecules are all helpful for enhancing the mechanical properties of materials, however, the ideal type and structure of shape memory polymer materials cannot be deduced according to the existing theory, which is a problem to be solved at present.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a shape memory polyurethane material and a self-reinforced regular pore polymer film prepared from the shape memory polyurethane material, and aims to provide a porous polymer film with mechanical properties and regular pores and use the porous polymer film for preparing an artificial periosteum.

An artificial periosteum is prepared by taking a porous polymer film as a raw material,

the porous polymer film is prepared by taking shape memory polyurethane as a raw material and adopting a water drop template method;

the shape memory polyurethane is an amorphous polymer with a glass transition temperature of 37-45 ℃ or a semi-crystalline polymer with a melting point of 37-45 ℃, and is prepared from diisocyanate, a soft-block polymer and

according to a molar ratio of 1.2-8: 1: 0.2-7, the molecular weight of which is 30000-150000;

the soft segment polymer is polylactic acid, polyglycolic acid, polycaprolactone, polylol or a copolymer of at least two of the polylactic acid, the polyglycolic acid, the polycaprolactone and the polylol.

Preferably, the soft segment polymer is represented by HO-R-OH, and the structural formula of the shape memory polyurethane is shown as formula I:

wherein x is selected from 1-10, y is selected from 1-10;

is a repeating unit of the soft segment polymer;

being repeat units of diisocyanates, or diisocyanates and

repeating units of the copolymer of (1).

Preferably, the molecular weight of the soft segment polymer is 1000-10000.

Preferably, the soft segment polymer is a polymer of lactic acid and a polyol.

Preferably, the molar ratio of the repeating unit of lactic acid to the repeating unit of polyol in the soft segment polymer is 10-102: 1-20.

Preferably, the soft segment polymer has a structural formula shown in formula II:

wherein m and n are respectively and independently selected from 4 to 50, and r is selected from 1 to 20.

Preferably, the diisocyanate is selected from aliphatic diisocyanates including hexamethylene diisocyanate, lysine diisocyanate, isophorone diisocyanate, dicyclohexylmethane diisocyanate, or aromatic diisocyanates including at least one of toluene diisocyanate or diphenylmethane diisocyanate.

Preferably, the

Comprises the following steps:

preferably, the parameters of the water drop template method are selected as follows: the environmental temperature is 4-50 ℃, the humidity is 25-80%, and the volatilization rate of the solvent is 5-50 mu L/min.

The invention also provides application of the porous polymer film in preparing an artificial periosteum.

The invention also provides a preparation method of the porous polymer film, which comprises the following steps:

step 1, dissolving the shape memory polyurethane in a volatile solvent to obtain a polyurethane solution;

and 2, coating the polyurethane solution on the surface of a substrate, and volatilizing in humid air to obtain the polyurethane coating.

Preferably, in step 1, the volatile solvent is at least one selected from chloroform, dichloromethane, tetrahydrofuran or carbon disulfide;

and/or the concentration of the polyurethane solution is 5-200 mg/mL;

and/or, in the step 2, the substrate coated with the polyurethane solution is dried under the conditions of the ambient temperature of 4-50 ℃ and the humidity of 25-80%, and the volatilization rate of the solvent is 5-50 mu L/min.

The invention designs a novel shape memory polyurethane material, and after a porous polymer film with regular pores is prepared by using a water drop template method, the microstructure of the film material shows certain crystal characteristics. This indicates that the molecular chains in the porous polymer film are arranged more orderly and the hard segment assembly is better, which makes the prepared porous polymer film have good mechanical properties. Compared with the similar porous polymer film, the high-mechanical-property porous polymer film provided by the invention has a good application prospect in the fields of artificial periosteum and the like with higher requirements on the mechanical property of the film.

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 shows two coupling agents of example 1¹H NMR spectrum;

FIG. 2 is an FT-IR spectrum of two coupling agents of example 1;

FIG. 3 is a FT-IR spectrum of PDLLA-PEG400-PDLLA macrodiol and ISO-PUs of example 1 (right spectrum is an enlargement of the rectangular marked part of the left spectrum);

FIG. 4 shows the PDLLA-PEG400-PDLLA and ISO-PUs of example 1¹H NMR spectrum;

FIG. 5 is a GPC curve of ISO-PUs in example 1;

fig. 6 shows the macroscopic morphology (thickness 0.032 ± 0.011mm) of ISO-PUs porous film (a) and non-porous control film (b);

FIG. 7 is an optical microscope image of condensation of water droplets on the surface of an ISO-PUs solution during formation of a porous membrane;

FIG. 8 is an SEM image of ISO-PUs porous membrane after drying, (b) is an enlargement of the phase red region in (a);

FIG. 9 is an optical microscope image of an ISO-PUs porous film (a) and optical microscope images under crossed polarizers (b-d), (c) and (d) are enlargements of the red region in (b);

FIG. 10 is an optical microscope image and a microscope image under cross-polarizer of an ISO-PUs porous membrane prepared at rates of 10. mu.l/min ((a) and (b)) and 40. mu.l/min ((d) and (e)) ((c) and (f) are magnified images of the red region in (b) and (e), respectively);

FIG. 11 is the microscopic morphology under optical microscope and cross-polarizer of porous membranes of PPZ-PU (a and b), Upy-PU (c and d), and PDLLA (e and f);

FIG. 12 is a DSC plot of an ISO-PUs non-porous control film and an ISO-PUs porous film (where (a) is the first heat cycle plot and (b) is the cool down and second heat curves);

FIG. 13 is a 2D-WAXD diffraction pattern for ISO-PUs nonporous control film (a) and ISO-PUs porous film (b), and a 1D-WAXD curve for ISO-PUs nonporous control film (c) and ISO-PUs porous film (D);

FIG. 14 shows FT-IR spectra (a) and 3260 and 3460cm of ISO-PUs control thin and porous membranes^-1Fitting a peak separating curve (b) of the region;

FIG. 15 is a tensile stress-strain curve (a) and mechanical properties (b) at 25 ℃ for ISO-PUs porous and nonporous transparent control films;

FIG. 16 is the cell morphology of rMSCs after 24h of culture on ISO-PUS nonporous control membranes (a and c) and porous membranes (b and d);

FIG. 17 is a plot of the spread area and aspect ratio of cells after 24h incubation of rMSCs on ISO-PUS nonporous control and porous membranes;

FIG. 18 shows the results of alkaline phosphatase (ALP) staining experiments after 14 days of incubation of rMSCs on ISO-PUS nonporous control membranes and porous membranes.

Detailed Description

Reagents and materials not specifically described in the following examples and experimental examples are commercially available.

Example 1 shape memory polyurethane

The present embodiment provides a shape memory polyurethane material. The preparation method comprises the following steps:

1. synthesis and purification of PDLLA-PEG400-PDLLA macrodiol

Mixing D, L-Lactide (D, L-Lactide, melting point: 118 deg.C, purity: 99.9%), PEG400 (Aladdin, CAS No. 25322-68-3, cat No. P103723), Sn (Oct)₂(Sigma-Aldrich, CAS number: 301-10-0, cat number: S3252) in a molar ratio of 5000:100:1 was added to a round-bottomed flask equipped with a magnetic stirrer, and the flask was evacuated for 30min and then sealed; putting the single-mouth bottle into an oil bath kettle 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 ℃) coprecipitation system is adopted to repeatedly purify the product for three times, and then the dichloromethane/ice normal hexane (-15 ℃) coprecipitation system is used to purify the product for one time; vacuum drying at room temperature for 72h to obtain target product PDLLA-PEG400-PDLLA macrodiol (m is 46, n is 46 and r is 10 in the structural formula) for use.

2. Synthesis and purification of HDI blocked ISO novel diisocyanate

HDI (Aladdin, CAS number: 822-06-0, cat number: H106723) and ISO (

Sigma-Aldrich, CAS number: 652-67-5, cargo number: i157515) were added to different round-bottom single-neck flasks, respectively, in a molar ratio of 4:1, while a magnetic stirrer was placed, in m (ISO, g): adding anhydrous DMF into two single-mouth bottles at a ratio of 1:6 (DMF, mL), and magnetically stirring to completely dissolve ISO; further mixing with Sn (Oct)₂Adding Sn (Oct) in a molar ratio of 500:1₂After three times of nitrogen replacement, reacting for 1h at 75 ℃ under the protection of nitrogen; after the reaction is finished, cooling to room temperature, pouring into n-hexane dried by a molecular sieve for precipitation to obtain white powder, drying to constant weight, namely the novel diisocyanate coupling agent of the HDI blocking ISO (in the structural formula in the embodiment, the value of z is 0), and reserving for later use.

3. Synthesis and purification of ISO-PUs

Wherein, OCN-DI' -NCO is the novel diisocyanate synthesized in the step 2. Wherein y is 5.

OCN-DI' -NCO and PDLLA-PEG400-PDLLA (macrodiol) were added in a molar ratio of 1.5:1.0 to a round-bottomed four-necked flask equipped with mechanical stirring and thermometer, while mixing in m (macrodiol, g): adding anhydrous DMF (DMF) according to the proportion of 1.0:0.8, and mechanically stirring to completely dissolve the macrodiol; then, the macrodiol and Sn (Oct)₂Adding Sn (Oct) in a molar ratio of 500:1₂Reacting for 6h at 75 ℃ under the protection of nitrogen (20 vol% of anhydrous DMF is added into the reaction system in each 2h to reduce the viscosity of the system); adding ISO at a ratio of 0.5:1.0 of ISO to macroglycol, and continuing to react for 12h at 75 ℃ under the protection of nitrogen (20 vol% of anhydrous DMF in the initial volume is added into the reaction system every 4h to reduce the viscosity of the system). And cooling to room temperature after the reaction is finished, pouring the reaction system into normal-temperature absolute ethyl alcohol for precipitation, and separating out white solid, namely ISO-PUs. And finally, purifying the ISO-PUs twice by using a dichloromethane/absolute ethyl alcohol coprecipitation system, and drying for later use. The glass transition temperature of ISO-Pus was determined to be 42 ℃.

4. Structural characterization

4.1 structural characterization of the coupling agent

FIGS. 1(a) and (b) new diisocyanate nuclear magnetic hydrogen spectra of sample HDI-blocked ISO obtained by HDI and ISO reactions at 4:1 and 2:1, respectively (HDI-blocked ISO¹H NMR) spectrum, the proton absorption peak positions on the two lines are consistent, but the intensities are greatly different. Wherein peaks at σ ═ 3.98 to 4.29(H-1, H-6), σ ═ 4.78(H-3), σ ═ 5.12(H-4), and σ ═ 5.28 to 5.32(H-2, H-5) are proton absorption peaks in ISO; peaks at σ ═ 1.30 to 1.48(H-c ', H-c), σ ═ 1.58 to 1.72(H-b ', H-b) and σ ═ 3.14 to 3.29(H-a ', H-a) belong to methylene proton absorption peaks in HDI; of two products¹No unreacted proton adsorption on the ISO ring appeared in the H NMR spectrumA peak was taken indicating complete capping of ISO by HDI. According to the theoretical formula (HDI-ISO (-HDI-ISO)_zHDI) it can be seen that the molar ratio of HDI to ISO in the two coupling agents can be calculated from the integrated areas of H-a', a and peak H-3, as shown in formula (4-1):

HDI/ISO＝I_H-a′+a/(I_H-3×4) (4-1)

for FIG. 1(a), HDI/ISO was close to 2 (7.74/(1.00X 4) ≈ 2), indicating that HDI and ISO gave HDI-ISO-HDI in a 4:1 reaction. While in FIG. 1(b) HDI/ISO is close to 1.5 (6.37/(1.00X 4) ≈ 1.5), indicating that HDI and ISO resulted in a 2:1 reaction of HDI-ISO-HDI-ISO-HDI.

Furthermore, in the FT-IR spectra of the two coupling agents (FIG. 2) it can be clearly observed that the peak intensity is 2273cm^-1An absorption peak of-NCO group appeared, and at 1698cm^-1An absorption peak of-C ═ O group in the urethane bond appeared. And 1698cm in HDI-ISO-HDI-ISO-HDI spectrogram^-1And 2273cm^-1The peak area of the position is obviously larger than the ratio of the two peak areas in the HDI-ISO-HDI spectrogram. These all further demonstrate the successful synthesis of the novel diisocyanates HDI-ISO-HDI and HDI-ISO-HDI-ISO-HDI.

4.2 ISO-PUs structural characterization

The samples synthesized with HDI as coupling agent were designated ISO1-PU, while the samples synthesized with HDI-ISO-HDI and HDI-ISO-HDI-ISO-HDI as coupling agent were designated ISO2-PU and ISO3-PU, respectively. The FT-IR spectra of PDLLA-PEG400-PDLLA and ISO-PUs are shown in FIG. 3. 1751cm in the PDLLA-PEG400-PDLLA curve^-1The absorption peak is the absorption peak of ester-C ═ O in PDLLA-PEG 400-PDLLA. No observation at 2273cm was made in all ISO-PUs spectra^-1The peak of-NCO absorption appeared, and some new peaks appeared compared with the spectrum of PDLLA-PEG 400-PDLLA. Namely 3300-^-1、～1621cm^-1And-1529 cm^-1The peaks respectively belong to the stretching vibration absorption peaks of-NH, an amide I band (-C ═ O) and an amide II band (-NH) in the urethane bond. In conclusion, the occurrence of these absorption peaks demonstrates the successful synthesis of ISO-PUs.

To further explore the molecular structure of ISO-PUs, PDLLA-PEG400-PDLLA and ISO-PUs were also performedTo master¹The obtained spectrum is shown in FIG. 4 by H NMR measurement. In PDLLA-PEG400-PDLLA¹In the H NMR spectrum, peaks at σ ═ 1.57ppm (H-B) and σ ═ 1.48ppm (H-B') were assigned to-CH groups of lactoyl units in macrodiols, respectively₃and-CH in the terminal lactic acid residue of the macroalcohol₃A proton absorption peak; the peak at σ ═ 3.64ppm (H-D) was from-CH within PEG400₂A proton absorption peak; peak σ ═ 5.16ppm (H-a) is the-CH proton absorption peak on the macrodiol lactoyl units; the peaks at sigma-4.23-4.46 ppm (H-A', H-C) are-CH linked to-OH on the terminal lactic acid residue of the macrogol and-CH adjacent to PDLLA block in PEG400₂The proton absorption peak of (1). Using a peak σ of 5.16ppm (I)_H-A17.08) and σ 4.23 to 4.36ppm (I)_H-A'+CIntegrated value of 1.00) and expression (4-2), and the molecular weight (M) of PDLLA-PEG400-PDLLA used was calculated_n) Is 7700.

Three ISO-Pus combinations¹Proton absorption peaks of-CH and-CH 3(H-a ', H-B ') attached to — OH at terminal lactic acid residue of PDLLA-PEG400-PDLLA at spectrum σ 4.36 to 4.46ppm and σ 1.48ppm in H NMR almost completely disappeared, and proton absorption peaks belonging to H-a ', H-B ', and H-c ' of HDI appeared at σ 3.14ppm, σ 1.38ppm, and σ 1.28 ppm. Furthermore, the characteristic proton peaks on the ISO ring (H-1 to H-6) are evident in the spectra of ISO2-PU and ISO3-PU, whereas they cannot be observed in the spectrum of ISO1-PU due to the low ISO content. The proton absorption peak at 3.64ppm assigned to H-D in PDLLA-PEG400-PDLLA was used as an internal standard (I)_H-D1.00) while taking the integrated area of the H-3 peak (I)_H-3) Or the integrated area of the H-4 peak (I)_H-4) Represents the ISO content, in terms of the integral value (I) of the H-a' peak_H-a') Representing the HDI content, the actual ratio of PDLLA-PEG400-PDLLA/ISO/HDI in ISO-PUs can be calculated, and the calculation results are shown in Table 1. M of ISO-PUs derived from GPC measurement (FIG. 5) at the same time_wAnd PDI are also listed in table 1. The difference between the actual ratio of PDLLA-PEG400-PDLLA/ISO/HDI and the theoretical ratio is larger as the molecular length of the coupling agent is increasedMeanwhile, the lower the molecular weight of PU is, this is caused by the difference in reactivity of the coupling agent.

TABLE 1 composition ratio and molecular weight data for three ISO-PUs

The above test results show that ISO-PUs have been successfully synthesized and that their molecular and theoretical structures have been consistent.

Example 2 high mechanical Property porous Polymer film with regular porosity

This example prepares the shape memory polyurethane material of example 1 into a porous polymer film with high mechanical properties and regular pores by the following method:

the ISO-PUs prepared in example 1 (ISO2-PU, M) were weighed out_w61kDa and PDI 1.57), the solution was completely dissolved in chloroform in a beaker, and the volume was determined in a volumetric flask to 50 mg/mL. And (5) carrying out three times of twice steaming and water washing on the cover glass, and carrying out twice of absolute ethyl alcohol washing and then airing. The coverslip was placed on a horizontal laboratory bench in a fume hood at an ambient temperature of 28 ℃ and a humidity of 70%, and about 200. mu.L of ISO-PUs solution was applied uniformly to the surface of the coverslip using a pipette gun while adjusting the air flow rate to a volatilization rate of about 20. mu.L/min. And after the solvent is completely volatilized, the porous film with the regular hexagonal pore structure can be obtained. The pore diameter of the porous film can be adjusted by controlling the volatilization rate, and the faster the volatilization rate of the solution is, the smaller the pore diameter is, and vice versa. By utilizing the principle, porous films with different pore diameters are prepared at 10 mu L/min and 40 mu L/min respectively.

Comparative examples 1 PPZ-PU, Upy-PU and PDLLA cellular porous films

Comparative example PPZ-PU (M) was prepared by the method of example 2_w＝80kDa,PDI＝1.66)、Upy-PU(M_w25kDa, PDI 2.12) and PDLLA (M)_w75kDa, PDI 1.35) were made into honeycomb porous membranes. Wherein, the synthesis process of PPZ-PU and Upy-PU is as follows:

wherein the R group of PPZ-PU is:

the R group of Upy-PU is:

HDI and PDLLA-PEG400-PDLLA (macrodiol) were added in a molar ratio of 1.5:1.0 to a round-bottomed four-necked flask equipped with mechanical stirring and a thermometer while mixing in m (macrodiol, g): adding anhydrous toluene (Tol) according to the proportion of 1.0:4.0 in V (DMF, mL), and mechanically stirring to completely dissolve the macrodiol; then, the macrodiol and Sn (Oct)₂Adding Sn (Oct) in a molar ratio of 500:1₂Reacting for 3 hours at 75 ℃ under the protection of nitrogen; then PPZ or Upy is added according to the molar ratio of PPZ or Upy to the macroglycol of 0.5:1.0, and the reaction is continued for 6h at 75 ℃ under the protection of nitrogen. And cooling to room temperature after the reaction is finished, pouring the reaction system into normal-temperature absolute ethyl alcohol for precipitation, and separating out white solid, namely PPZ-PU or Upy-PU. And finally, purifying the PPZ-PU or Upy-PU twice by using a dichloromethane/absolute ethyl alcohol coprecipitation system, and drying for later use.

The synthesis of PDLLA is as follows:

mixing D, L-Lactide (D, L-Lactide, melting point: 118 ℃, purity: 99.9%), Sn (Oct)₂(Sigma-Aldrich, CAS number: 301-10-0, cat number: S3252) in a molar ratio of 5000:1, placing in a round-bottomed flask with a magnetic stirrer, vacuumizing for 30min, and sealing; putting the single-mouth bottle into an oil bath kettle at 140 ℃, starting magnetic stirring after the mixture is completely melted to uniformly stir the reaction system, and continuously reacting for 14 hours; after the reaction is finished, a dichloromethane/normal temperature absolute ethyl alcohol coprecipitation system is adopted to repeatedly purify the product for three times, and vacuum drying is carried out for 72 hours at room temperature, thus obtaining the target product PDLLAAnd then is ready for use.

COMPARATIVE EXAMPLE 2 ISO-PUs NON-POROUS CONTROL FILM

This comparative example, which was prepared using a similar procedure to example 2 for the preparation of an ISO-PUs nonporous control film, differs from example 2 in that: the humidity was set at 30%, and the air flow rate was adjusted to give a volatilization rate of 20. mu.L/min.

The advantageous effects of the present invention will be further described by experiments.

Experimental example 1 structural characterization

The samples used in this experimental example were the ISO-PUs porous polymer film prepared in example 2, the PPZ-PU, Upy-PU and PDLLA cellular porous films prepared in comparative example 1, and the ISO-PUs non-porous control film prepared in comparative example 2.

First, experiment method

Testing by a polarizing microscope: after the porous film on the cover glass is completely dried, the glass slide is placed on an MP41 type polarizing microscope (Bright and American photoelectric) stage, the appearance of the porous film under normal transmission light and orthogonal polarization at the corresponding position is respectively observed in a high power lens (20-50 times) transmission mode, and is recorded by a digital camera.

Scanning Electron Microscope (SEM) test: the completely dried porous film sample was removed from the cover glass, and SEM test was performed on the film surface using a JSM-7800F scanning electron microscope (Nippon electronics Co., Ltd.). Before testing, the sample is sprayed with gold, the thickness is about 0.5nm, and the testing voltage is 8 KV. The C, O and N elements of the sample were imaged simultaneously using x-ray electron spectroscopy (EDS).

Testing one-dimensional wide-angle X-ray diffraction (1D-WAXD): the film samples were subjected to a 1D-WAXD test using a panalyticic-Empyrean high resolution X-ray diffractometer (marvens parnake, netherlands) with a Cu target scan rate 2 θ of 1 °/min over a scan range of 5 ° -35 °.

And (2D-WAXD) test: the film was subjected to two-dimensional wide-angle x-ray diffraction (2D-WAXD) analysis at the BL16B1 beam line station of the Shanghai synchrotron radiation apparatus (SSRF). The sample was at a distance of 30mm from the detector and the diffraction pattern was acquired by a Pilatus300K detector with a resolution of 487 x 619 pixels with a test time of 45 s.

FT-IR test: fourier transform infrared (FT-IR) spectra of film samples were collected using an IRTracer-100 attenuated reflectance FT-IR spectrometer (Shimadzu, Japan). Each sample was scanned 16 times with a resolution of 4cm^-1Recording the sample at 4000-700 cm^-1In-range spectra. The spectra were peaked and integrated using OriginPro 9.0 software after the test was completed.

Sixthly, DSC test: using a DSC 200F3 model (Netzsch, Germany) Differential Scanning Calorimeter (DSC) at N₂In the atmosphere, the temperature rise (decrease) rate is 10 ℃/min. The sample was first heated from-10 ℃ to 150 ℃, then cooled to-10 ℃ and finally heated again to 150 ℃. Data for the first temperature rise, temperature fall, and second temperature rise cycles are recorded.

Second, experimental results

1. Macroscopic morphology of ISO-PUs porous film

FIG. 1 is a comparison photograph of the macro-topography of ISO-PUs porous films and non-porous control films. The porous membrane (fig. 6(a)) is translucent without forming a complete porous structure visually except for the edge region where the solution is volatilized too fast, and most of the region is white and opaque. All subsequent porous films were tested for visually white opaque areas. While the ISO-PUs non-porous film as a control (FIG. 6(b)) appeared colorless and transparent, indicating that no phase separation structure was present or did not exceed the nanometer scale in the non-porous control film.

2. Micro-morphology of ISO-PUs porous film

FIG. 7 is a photograph showing the condensed water droplets accumulated on the surface of the solution during the various film manufacturing processes of ISO-PUs (solvent evaporation rate of about 20. mu.L/min). It can be observed that the agglomerated water droplets (like white particles) are regularly arranged in most areas, the water droplets are in the shape of regular hexagons and there are distinct boundaries between the individual water droplets. This area appears as a single layer of regular hexagonal pore area in the SEM test (fig. 8(a)) after complete evaporation of the solvent and water. While in a small fraction of the area, the droplets are interconnected and overlap, and eventually complete evaporation of solvent and water appears as a multi-layered pore area (fig. 8 (a)). Fig. 3 is a photograph of the morphology obtained by SEM test after the honeycomb-shaped porous film was completely dried, the size of the pores was very uniform, and each of the regular hexagonal pores had a diameter of about 4.0 μm (fig. 8 (b)).

When the porous film was observed in the transmission mode using a microscope, the single-layer hole was visually white, and the multi-layer hole was visually red due to the difference in light transmittance (fig. 9 (a)). When further observed under crossed polarisers (fig. 9(b)), an odd image can be observed in the single layer region, with the four edges symmetrically disposed on either side of the hexagon showing crystal-like features, and the two remaining edges lying between the four edges showing no crystal features, i.e. being visually completely dark. At the same time, the edges with crystalline character have a regio-orientation, and the boundaries of these differently oriented regions are the multi-layer pore regions. Further magnification of these regions revealed that each edge having a crystalline character was "Z" shaped, and that two adjacent crystalline edges in the same orientation region were not closely connected, and that a space of about 0.3 to 0.5 μm existed between them (FIGS. 9(c) and (d)).

Further, example 2 obtained ISO-PUs porous films having pore diameters of 1.5 μm and 7.0 μm by adjusting the air flow rate so that the solvent volatilization rates were 10 μ l/min and 40 μ l/min, respectively, and the optical microscopic morphologies of these two films and the microscopic morphology under crossed polarizers are shown in FIG. 10. When the optical microscope morphologies in FIGS. 10(a) and (d) are compared with the morphology of the porous film having a pore size of about 4.0 μm in FIG. 9(a), it can be seen that the pore size is proportional to the size of the multi-layer pore-forming region, i.e., the film having a larger pore size exhibits a larger multi-layer pore region area. The two hexagonal holes with diameters of 1.5 μm and 7.0 μm (FIGS. 10(b) and (e)) under crossed polarizers showed similar crystal morphologies to the hexagonal holes in FIG. 9 (b). However, due to the resolution of the optical microscope, "zigzag" crystal morphology at the edges of hexagonal holes with diameters of 1.5 μm became less pronounced (FIG. 10(c)), while "zigzag" crystal morphology at the edges of hexagonal holes with diameters of 7.0 μm became more pronounced (FIG. 10 (f)).

Comparative example 1 PPZ-PU, Upy-PU and PDLLA porous membranes were also prepared separately by BF method, wherein PPZ-PU and Upy-PU have the same PDLLA-PEG400-PDLLA soft segment molecular chain as ISO-PUs. FIG. 11 shows the microscopic morphology of these porous films under optical microscopy and crossed polarizers, and the porous films PPZ-PU (FIG. 11(a)) and UPy-PU (FIG. 11(c)) are also honeycomb-shaped, i.e., the surface pores are all regular hexagons and the diameter of the pores is similar to that of the ISO-PUs porous film in FIG. 9 (a). The pores on the surface of the PDLLA porous membrane are irregular, and the pore diameter is obviously smaller than that of PPZ-PU and Upy-PU. Importantly, the porous films of these three materials did not exhibit crystalline characteristics when viewed under a polarizer (fig. 11(b), (d) and (f)), i.e., were completely dark in the field of view. Since ISO-PUs differs from PPZ-PU (and Upy-PU) only in the presence of the repeating units of the bicyclic ISO small molecule, it can be speculated that the bicyclic ISO small molecule plays a crucial role in the formation of similar crystal structures in fig. 9 and 10.

3. Thermal performance of ISO-PUs porous films

To further confirm the presence of crystals on the edges of the ISO-PUs porous membranes, DSC tests were performed on ISO-PUs porous membranes and ISO-PUs non-porous control films having a diameter of 4.0 μm. The first thermal scan curve is shown in FIG. 12(a), and the DSC curve of the porous film has a typical crystal melting peak, a melting range of 46.5-55.7 ℃, a melting point of 51.7 ℃, and a melting enthalpy of 9.97J/g, while the DSC curve of the control film has no similar melting peak. In addition, the glass transition temperature of the porous film was 43.1 ℃ which is much higher than 31.4 ℃ of the control film, probably due to the restriction of the molecular chains in the amorphous region by the crystals, indicating that there is a difference in the degree and structure of separation between the soft and hard phases in the two films.

The cooling curve and the reheating curve of both films were not significantly different (FIG. 12(b)), and neither of them exhibited a crystallization peak in the cooling curve nor a crystal melting peak in the reheating curve. In addition, the glass transition temperatures of the two films on the cooling curve and the secondary heating curve are substantially the same. It is stated that ISO-PUs are indeed an amorphous polymer, the crystals in the porous film being formed by the BF process.

4. WAXD analysis of ISO-PUs porous membranes

In order to further examine the crystal structure in the ISO-PUs porous film, a porous film having a pore size of 4.0 μm was subjected to two-dimensional wide-angle X-ray diffraction (2D-WAXD) and one-dimensional wide-angle X-ray diffraction (1D-WAXD) tests. The 2D-WAXD diffraction pattern and 1D-WAXD curve obtained using ISO-PUs non-porous clear films as control samples were also tested as shown in FIG. 13. In the 2D-WAXD diffraction pattern (fig. 13(a)) of the ISO-PUs control film, only a strong diffraction spot appears on the equator, and in the corresponding 1D-WAXD diffraction curve (fig. 13(c)), a sharp diffraction peak is evident at 9.8 ° 2 θ in addition to a broad diffraction peak of an amorphous phase. These results show that the molecular chains in the ISO-PUs control films spontaneously assemble to form structures on the nanometer scale and with an ultrahigh degree of orientation. However, the content of such ordered nanostructures was too small to observe a distinct melting peak in the DSC curve thereof (fig. 12 (a)). The 2D-WAXD diffraction pattern of the ISO-PUs porous film (FIG. 13(b)) is significantly different from the control film, with 5 typical elliptical diffraction rings in the pattern, corresponding exactly to 5 crystalline diffraction peaks in the 1D-WAXD diffraction curve (FIG. 13 (D)). These phenomena further verify that typical crystal structures exist in the porous film and that the crystal structures have a certain orientation.

5. FT-IR analysis of ISO-PUs porous films

The FT-IR spectrum of the total reflection of the ISO-PUs nonporous transparent control film and the cellular porous film with a pore size of 4.0 μm is shown in FIG. 14 (a). At 1528cm^-1The band (-NH group) of amide II in the amide bond can be seen at 1590-1870 cm^-1Absorption peaks were observed for the ester-C ═ O group in the macrodiol and the-C ═ O group in the amide bond. 3260-Astro 3460cm due to the partial overlap of the two types of-C ═ O groups^-1the-NH group at (A) is more suitable for characterizing the formation of hydrogen bonds. 3260 and 3460cm^-1The results of fitting the FT-IR spectrum of the region were shown in FIG. 14(b), and the free-NH groups in both films appeared at 3408cm^-1But they differ in the position where they form ordered and disordered hydrogen bond-NH groups. In the spectrum of the control film, an absorption peak at 3357cm was observed in which disordered hydrogen bond-NH groups were formed^-1Here, the absorption peak at which the ordered hydrogen bond-NH group was formed appeared at 3323cm^-1To (3). However, the absorption peaks for disordered and ordered hydrogen bond-NH groups formed in the porous film appeared at lower wave numbers than the corresponding-NH groups in the control film, which were shifted separatelyMoved to 3350cm^-1And 3315cm^-1To (3). The integral areas of peaks corresponding to-NH groups of different states in two sample curves obtained by peak splitting fitting are shown in table 2, the integral areas of peaks corresponding to ordered and unordered hydrogen bond-NH groups in the porous membrane are far larger than those of a control film, and the peak areas corresponding to free-NH groups are far smaller than those of the control film. This indicates that the molecular chains in the ISO-PUs porous films are significantly more oriented than the control films.

TABLE 2 fitting peak and integral calculations of-NH groups in ISO-PUs control and porous membranes

In summary, the characterization results of the experimental examples show that the shape memory polyurethane material provided by the invention can be combined with a water drop template method to prepare a porous polymer film containing an ordered crystal structure.

Experimental example 2 characterization of mechanical Properties

The samples used in this experimental example were the ISO-PUs porous polymer film prepared in example 2 and the ISO-PUs non-porous control film prepared in comparative example 2.

First, experiment method

Mechanical Properties of the samples measured in ElectroPuls with an optical extensometer^TMTensile mechanical testing was performed on an E1000 test instrument (Instron, USA) at a loading rate of 5.0 mm/min. The samples were thin strips (thickness about 32 μm, width 10mm, full length 50mm, gauge length 25 mm). The final result is the average of 5 replicates.

Second, experimental results

The phase structure of the polymer has an important influence on its mechanical properties. Thus, the tensile mechanical properties of ISO-PUs cellular porous membranes having pore diameters of 4.0 μm were evaluated in this chapter and, likewise, using a nonporous, transparent ISO-PUs film as a control, the thicknesses of the two films were very close. The obtained stress-strain curve and the mechanical properties calculated according to the stress-strain curve are shown in fig. 15(a) and (b), the porous film obviously has better mechanical properties than the reference film, and the calculated tensile strengths of the porous film and the reference film are respectively 21.5 +/-4.1 MPa and 16.3 +/-1.8 MPa; the Young modulus is 942.9 +/-111.7 MPa and 759.9 +/-99.7 MPa respectively; the elongation at break is 31.6 + -3.2% and 11.4 + -2.7%, respectively.

Therefore, compared with a control film with the thickness close to the same polymer raw material, the porous polymer film prepared by the method has obviously improved mechanical properties. And according to the above characterization results of mechanical properties, the porous polymer film of the present invention has reached the mechanical property requirements of artificial periosteum (tensile strength 3-4 MPa, elongation at break 19.6-34.8%, from biomech, 2003,18:760-764) and anti-adhesion membrane (Young's modulus 131.4MPa, tensile strength 9.5MPa, elongation at break 630%, Materials Science and Engineering: C,2020,117: 111283).

Experimental example 3 Effect of porous Structure on morphology and osteogenic differentiation of rMSCs

First, experiment method

The ISO-PUs porous and non-porous control films were vacuum dried at 40 ℃ for 7 days to completely remove the chloroform. After ultraviolet irradiation for 30min, both sides of the sample were placed in a 24-well plate (material film side up), and rat bone marrow mesenchymal stem cells (rMSCs) were seeded at a cell density of 0.6X 104/cm 2. After co-culturing for 24h in high-sugar medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, the medium was aspirated from the 24-well plate and washed 3 times with PBS. Then fixing the rMSCs by using an immunostaining fixing solution at room temperature for 2H, respectively staining cell nuclei and skeletons by using H33258 and phalloidin, and observing the forms of the rMSCs by using a TCS SP8 laser confocal microscope. Aspect ratio and spread area of rMSCs were statistically analyzed using ImageJ software. After 14 days of co-culture, the medium was aspirated from the 24-well plate and washed 3 times with PBS; then fixing for 2 hours at room temperature by using an immunostaining fixing liquid; then staining the kit with alkaline phosphatase (ALP) for 2h at room temperature; finally, the staining of the sample was observed and recorded by a light microscope (MP41, Mingmei photoelectric) with a camera.

Second, experimental results

The cell morphology of rMSCs after 24h culture on ISO-PUS nonporous control membranes and cellular porous membranes with pore size of-4.0 μm is shown in FIG. 16. There was a clear difference in cell morphology between the two film surfaces, with the mscs on the surface of the ISO-PUs non-porous control film being mostly triangular or quadrilateral and a small part being circular (fig. 16(a) and (c)), while the mscs on the surface of the ISO-PUs porous film were almost all elongated quadrilateral (fig. 16(b) and (d)), and it was clearly seen that the cellular pseudopodia adhered to the edges of the hexagonal wells. The present study also counted the spread area and aspect ratio distribution of the rMSCs on both films, and the resulting distribution is shown in fig. 17. The graph shows that the rMSCs on the surfaces of 116 ISO-PUs porous films and 105 ISO-PUs non-porous control films are counted respectively, the spreading areas of the rMSCs on the two films are similar overall, and only a few rMSCs on the surfaces of the porous films have the spreading areas which are far larger than those on the surfaces of the rMSCs on the surfaces of the control films. Meanwhile, the distribution of the length-diameter ratio of the rMSCs on the surface of the comparison film is concentrated, the distribution of the length-diameter ratio of the rMSCs on the surface of the porous film is dispersed, and the length-diameter ratio of the rMSCs is generally larger than that of the rMSCs on the surface of the comparison film. ALP is an early osteogenic marker that directly reflects the activity or functional status of osteoblasts. It can be seen from fig. 18 that the cells on both the control film and the cellular porous film after staining are dark blue, and the cells on the cellular porous film are darker in color, i.e., higher in ALP content. The results show that the rMSCs are subjected to osteogenic differentiation on the two films, but the rMSCs on the cellular porous film are more obviously osteogenic differentiation, and the osteogenic activity is higher after the rMSCs are differentiated into osteoblasts.

The above results indicate that the ISO-PUs porous membranes have a better promoting effect on osteogenic differentiation of the rMSCs compared to the non-porous control membranes. The porous polymer film provided by the invention has application potential as an artificial periosteum.

As can be seen from the above examples and experimental examples, the present invention designs a new shape memory polyurethane material, and combines the shape memory polyurethane material with a water drop templating method, and can comprise a porous polymer film having a crystalline structure. The porous film material has excellent mechanical properties, can meet the performance requirements of application scenes such as artificial periosteum and the like, and has good application prospects.

Claims

1. An artificial periosteum, comprising: it is made up by using porous polymer film as raw material,

according to a molar ratio of 1.2-8: 1: 0.2-7, and the molecular weight is 30000-150000;

2. The artificial periostin of claim 1, wherein: the soft segment polymer is represented by HO-R-OH, and the structural formula of the shape memory polyurethane is shown as formula I:

wherein x is selected from 1-10, y is selected from 1-10;

is a repeating unit of the soft segment polymer;

being repeat units of diisocyanates, or diisocyanates and

repeating units of the copolymer of (1).

3. An artificial periostin according to claim 1 or 2, characterised in that: the molecular weight of the soft segment polymer is 1000-10000.

4. An artificial periostin according to claim 1 or 2, characterised in that: the soft segment polymer is a polymer of lactic acid and polyalcohol.

5. The artificial periostin of claim 4, wherein: the molar ratio of the repeating unit of the lactic acid to the repeating unit of the polyalcohol in the soft segment polymer is 10-102: 1-20.

6. The artificial periostin of claim 5, wherein: the structural formula of the soft segment polymer is shown as a formula II:

7. An artificial periostin according to claim 1 or 2, characterised in that: the diisocyanate is selected from aliphatic diisocyanate or aromatic diisocyanate, the aliphatic diisocyanate comprises hexamethylene diisocyanate, lysine diisocyanate, isophorone diisocyanate and dicyclohexylmethane diisocyanate, and the aromatic diisocyanate comprises at least one of toluene diisocyanate or diphenylmethane diisocyanate.

8. The artificial periostin of claim 7, wherein: the above-mentioned

Comprises the following steps:

9. the artificial periostin of claim 1, wherein: the parameters of the water drop template method are selected as follows: the environmental temperature is 4-50 ℃, the humidity is 25-80%, and the volatilization rate of the solvent is 5-50 mu L/min.

10. Use of a porous polymer film with regular pores for the preparation of an artificial periosteum, characterized in that: the porous polymer film is prepared by taking shape memory polyurethane as a raw material and adopting a water drop template method;

the shape memory polyurethane is prepared from diisocyanate, soft segment polymer and