CN109970998B

CN109970998B - Method for preparing GelMA macroporous hydrogel by Pickering emulsion method and application

Info

Publication number: CN109970998B
Application number: CN201910194710.5A
Authority: CN
Inventors: 曹晓东; 潘昊田; 李庆涛
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2019-03-14
Filing date: 2019-03-14
Publication date: 2020-04-28
Anticipated expiration: 2039-03-14
Also published as: CN109970998A

Abstract

The invention discloses a method and application for preparing GelMA macroporous hydrogel by a Pickering emulsion method. In the method, GelMA and PEG-4SH are dissolved in a PBS buffer and stirred; Ultrasonic; adding dodecane and photoinitiator, stirring vigorously with a high-speed homogenizer to obtain a stable Pickering emulsion; subjecting the emulsion to UV light to form a gel, and thoroughly washing and dialysis with ethanol and water to obtain GelMA macroporous hydrogel. The prepared GelMA macroporous hydrogel has the advantage of a macroporous structure, which can promote the transport of nutrients, the discharge of metabolites and the communication between cells; and the Mg-loaded component is effective for mouse bone marrow mesenchymal stem cells. It has the functions of promoting adhesion, proliferation and osteogenic differentiation, has excellent biocompatibility, can be used as a bone tissue engineering scaffold with excellent performance, and has great clinical application potential in the field of bone repair.

Description

Method for preparing GelMA macroporous hydrogel by Pickering emulsion method and application

Technical Field

The invention relates to the technical field of hydrogel materials, in particular to a method for preparing GelMA macroporous hydrogel by a Pickering emulsion method and application thereof.

Background

Bone injury is a common surgical disease in clinic, and currently, bone grafts adopted in clinical treatment mainly comprise autogenous bone, allogeneic bone and xenogeneic bone, and the three clinical bone grafts have advantages and disadvantages and are limited in source, so that a large amount of bone repair materials are required for clinical treatment every year. Various bone repair materials used clinically also have certain problems, such as poor bone conductivity of medical metal materials and mismatching with the elastic modulus of natural bone tissues of human bodies; the calcium phosphate cement has poor mechanical strength and cannot be used for repairing bearing bones; the degradation speed of the bioactive glass is not matched with the growth speed of bone tissues, so that the problems of over-slow growth speed of the bone tissues or incomplete repair of bone defects and the like are caused.

The bone tissue has complex components and contains various trace metal elements such as Mg, Sr, Zn, Fe, Cu and the like. In the research of bone repair materials, the doping of trace metal elements can improve the bone repair performance of the materials. Among various trace metal elements, Mg is involved in the physiological processes of bone tissue formation, bone metabolism, bone mineral crystallization and the like, and has the effect of promoting the adhesion, proliferation and osteogenic differentiation behaviors of osteoblasts.

The polymer hydrogel is a material system formed by combining a three-dimensional network formed by crosslinking hydrophilic polymers and water molecules. The materials commonly used for preparing the bone repair hydrogel comprise natural biomass macromolecules such as collagen, gelatin, silk fibroin, chitosan, sodium alginate, hyaluronic acid and the like. The hydrogel prepared from these natural polymers is similar to the extracellular matrix of the body in terms of components, has good biological properties and degradation properties, has the advantages of promoting the adhesion of cells on materials and the like, and is widely applied to the research of bone repair implants. At present, various preparation methods are available for preparing hydrogel scaffolds with various forms from natural biomass polymer materials.

The macroporous gel scaffold has great application value in the aspects of tissue engineering, biomedicine and the like, and the mutually communicated pore structures in the hydrogel can promote the transportation of nutrient substances, the discharge of metabolites and the communication among cells, and can be used as a good tissue engineering scaffold. Hydrogels with effective pore sizes in the range of 10nm to 10 μm are generally referred to as microporous hydrogels, and hydrogels with effective pore sizes greater than 10 μm are generally referred to as macroporous hydrogels. In the common method for preparing the biomedical macroporous scaffold at present, communicating holes cannot be formed, or extra chemicals are required to be introduced to increase toxicity, which is not favorable for the biocompatibility of materials. According to the principle and characteristics of preparing a macroporous hydrogel support material by a Pickering emulsion method, MgO nano particles are used as a stabilizer of the Pickering emulsion, and a GelMA macroporous hydrogel support is designed to be used as a bone repair support with excellent performance.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a method for preparing GelMA macroporous hydrogel by a Pickering emulsion method and application thereof, wherein the macroporous hydrogel prepared by the method has the advantages of a macroporous structure and can promote the transportation of nutrient substances, the discharge of metabolites and the communication among cells; and the Mg-loaded component has the effects of promoting adhesion and proliferation and promoting osteogenic differentiation on mouse bone marrow mesenchymal stem cells, has excellent biocompatibility, can be used as a good tissue engineering scaffold, and has huge clinical application potential in the field of bone repair.

In order to achieve the purpose, the technical scheme provided by the invention is as follows: a method for preparing GelMA macroporous hydrogel by a Pickering emulsion method comprises the steps of firstly, carrying out graft modification on a gelatin molecular chain by methacrylic anhydride to obtain methacrylated gelatin, namely GelMA, carrying out thiol-ene click reaction on double bonds on the gelatin under the catalysis of a photoinitiator I2959 and sulfydryl on PEG-4SH on four arms under the irradiation of UV light to quickly form hydrogel, adding an oil phase solution consisting of MgO nano particles and dodecane into a gel precursor solution, namely a water phase, preparing stable and uniform Pickering emulsion under the high-speed stirring of a homogenizer, then irradiating the gel by the UV light, washing dodecane oil drops serving as a template, and thus obtaining the GelMA macroporous hydrogel stabilizing agent taking the MgO nano particles as the Pickering emulsion.

The method comprises the following steps:

1) dissolving GelMA and PEG-4SH in PBS (1 x) buffer solution to obtain aqueous phase solution;

2) adding MgO nano particles into dodecane, stirring, and then performing ultrasonic dispersion to obtain an oil phase solution;

3) mixing the water phase and the oil phase solution, adding an I2959 photoinitiator, and stirring at a high speed by using a homogenizer to prepare a uniform and stable Pickering emulsion;

4) and injecting the emulsion into a PDMS (polydimethylsiloxane) mold, irradiating by using UV (ultraviolet), forming the emulsion into gel, demolding, dialyzing by using absolute ethyl alcohol and deionized water, and freeze-drying to obtain the GelMA macroporous hydrogel taking MgO nano particles as a Pickering emulsion stabilizer.

In the step 1), the substitution degree of GelMA is more than 70%, and the molecular weight of PEG is 2-20 kDa.

In the step 1), the mass ratio of GelMA to PEG-4SH is 1: 0.5-1.

In the step 2), the particle size of the MgO nano-particles is 20-100 nm.

In the step 2), the ultrasonic time is 10-60 min.

In the step 3), the addition amount of the I2959 photoinitiator is 0.01-0.1 wt%.

In the step 3), the high-speed stirring speed of the homogenizer is 1-2 ten thousand revolutions per minute, and the stirring time is 30-90 s.

In the step 4), the thickness of the PDMS mold is 2-5 mm, the aperture is 5-10 mm, and the UV illumination time is 30-90 s for each of the front side and the back side of the mold.

The GelMA macroporous hydrogel prepared by the method is used as a tissue engineering scaffold for bone repair.

Compared with the prior art, the invention has the following advantages and beneficial effects:

the GelMA macroporous hydrogel material obtained by the invention has good biocompatibility and degradability. the thiol-ene click reaction condition is mild, the gelling time is short, the cell adhesion of gelatin and the excellent physical and chemical properties of PEG are combined, the tissue engineering scaffold material which is uniform in component, good in cell compatibility, adjustable in chemical and mechanical properties is prepared, and the natural extracellular matrix is well simulated. The MgO nano particles are introduced to be used as a Pickering emulsion stabilizer, so that the material is endowed with a macroporous structure, the transportation of nutrient substances, the discharge of metabolites and the communication among cells are promoted, the mechanical strength of the material is effectively improved, and Mg²⁺The release of the hydrogel can effectively promote the proliferation and osteogenic differentiation capability of cells, and is expected to become an excellent bone repair hydrogel scaffold. Has great clinical application potential.

Drawings

FIG. 1 is a gel-forming mechanism diagram of GelMA macroporous hydrogel.

FIG. 2 shows GelMA with gelatin¹H NMR。

FIG. 3 is a photograph showing a contact angle test of MgO nanoparticles.

FIG. 4 is a graph of the compressive modulus of a GelMA macroporous hydrogel scaffold.

Figure 5a is the EDS energy spectrum (whole face scan) of the GelMA macroporous hydrogel scaffold.

Figure 5b is EDS energy spectrum (local scan) of GelMA macroporous hydrogel scaffold.

FIG. 6a is Mg of GelMA macroporous hydrogel scaffold²⁺One of the in vitro release profiles.

FIG. 6b is Mg of GelMA macroporous hydrogel scaffold²⁺In vitro release profile two.

Fig. 7a, 7b, 7c, and 7d are SEM photographs of GelMA macroporous hydrogel scaffolds with different MgO concentrations.

FIG. 8 is a graph of the proliferation of

cells

1,3, and 7 days after mBMSCs were plated on GelMA macroporous hydrogel scaffolds with different MgO concentrations.

FIG. 9 is a laser confocal photograph of Live/Dead staining of mBMSCs planted on GelMA macroporous hydrogel scaffolds of different MgO concentrations, cultured to day 7.

Detailed Description

Specific implementations of the present invention are further described below with reference to examples, but the embodiments of the present invention are not limited thereto.

Example 1 (modification and characterization of gelatin)

Modification of gelatin

The GelMA is obtained by modifying gelatin through methacrylic anhydride, and is grafted on a gelatin molecular chain, and a carbon-carbon double bond with high reaction activity is introduced on the gelatin molecular chain, so that the GelMA is favorable for carrying out thiol-ene click reaction with sulfydryl on a four-arm PEG-4SH molecule, and is crosslinked into a gel network under UV illumination. The gel forming mechanism of the GelMA macroporous hydrogel is shown in figure 1.

5g of gelatin was dissolved in 50mL of PBS (1X) buffer, and the mixture was stirred in a water bath at 60 ℃ until completely dissolved. 10mL of methacrylic anhydride was dropped into the stirred gelatin solution at a rate of 0.5mL/min, and reacted in a water bath at 50 ℃ for 3 hours. After cooling slightly, the reaction was diluted 5-fold with 40 ℃ PBS (1X) to terminate the reaction. Dialyzing with 12-14kDa dialysis bag for 7 days, dialyzing in 40 deg.C water bath, and lyophilizing to obtain product for storage at 4 deg.C.

II, 1H NMR characterization of gelatin modified product

The molecular structure of the gelatin was analyzed by nuclear magnetic resonance hydrogen spectroscopy (1H NMR) with GelMA, and the 1H NMR spectra of both molecules were shown in FIG. 2.

In FIG. 2, the upper line is GelMA, the lower line is gelatin, and it can be seen that the two nuclear magnetic curves are basically consistent, GelMA has two chemical shifts at 5.35ppm and 5.6ppm, i.e. the characteristic peaks of methacrylamide, indicating that methacrylic anhydride has been successfully modified on the main chain of gelatin.

Example 2 (contact Angle test of MgO nanoparticles)

The Pickering emulsion is an emulsion obtained by using ultrafine solid particles as an emulsifier. Assuming that the solid particles are spherical, the state of the particles in the oil-water phase, the surface energy between particle-water, particle-oil, water-oil is critical to the type of emulsion formation. If the contact angle theta between the particles and the water phase is less than 90 degrees, the wettability of the solid particles and the water phase is better, and the system is easy to form an oil-in-water (O/W) emulsion; if the contact angle θ between the particles and the aqueous phase is >90 °, i.e. the particles are more wettable to the oil phase, the system is prone to form water-in-oil (W/O) emulsions. Therefore, we can roughly judge the type of emulsion by the contact angle of the particles and water, and the hydrophilicity and hydrophobicity of the particles must be moderate, so that no stable emulsion can be obtained by either being too hydrophilic or too hydrophobic.

Dispersing MgO powder in absolute ethyl alcohol, performing ultrasonic treatment for 30min to fully and uniformly disperse the MgO powder, then dripping the MgO powder on a very thin glass wafer, after the ethanol is completely volatilized, dripping one drop of the MgO powder, uniformly coating the MgO powder on the glass wafer, forming a film, and then performing a contact angle test. The photograph of the contact angle test of the MgO nanoparticles is shown in FIG. 3. The average value of the contact angle test of the MgO nano particles is 36.1 degrees.

The measured results indicate that the MgO nanoparticles are hydrophilic inorganic particles and can form stable O/W emulsion.

Example 3 (compression Performance test of GelMA macroporous hydrogel scaffolds)

0.05g GelMA was dissolved in 0.25mL PBS solution (1X) and stirred until well dissolved. 0.25mL of a 0.2g/mL PEG-4SH (1 ten thousand molecular weight) solution was mixed with GelMA solution. Add 1.5mg nanometer MgO to 0.5mL dodecane, sonicate for 15 min. GelMA, PEG-4SH, dodecane, I2959 photoinitiator were mixed and stirred with a homogenizer at 1.6 ten thousand revolutions per minute for 60 s. The emulsion was transferred to a PDMS mold 5mm in thickness and 1cm in diameter and UV-irradiated for 60 seconds on each of the front and back sides. Finally, the GelMA macroporous hydrogel scaffold for the compression modulus test is prepared. The compression curves of the hydrogel scaffolds with MgO contents of 0.3 wt% and 0.5 wt% are shown in FIG. 4.

The content of MgO in the 0.5 wt% group is higher, the particle size of emulsion droplets is smaller, so the aperture of the hydrogel is smaller, and the sample with high MgO content has higher compression modulus, thereby conforming to the general rule. Calculated from the slope of the first 20% deformation region in the graph, the compressive modulus for the 0.3 wt% MgO content sample was 6.99 kPa; the compressive modulus of the sample with 0.5 wt% MgO content is 12.33 kPa.

Example 4 (elemental analysis of GelMA macroporous hydrogel scaffolds)

0.1g GelMA was dissolved in 0.25mL PBS solution (1X) and stirred until well dissolved. 0.25mL of 0.2g/mL PEG-4SH (8 thousandths of a molecule) solution was mixed with GelMA solution. 2.5mg of nano MgO is added into 0.5mL of dodecane and ultrasonic treatment is carried out for 20 min. GelMA, PEG-4SH, dodecane, I2959 photoinitiator were mixed and stirred with a homogenizer at 2 ten thousand revolutions per minute for 60 s. The emulsion was transferred to a PDMS mold 2mm in thickness and 8mm in diameter and UV-illuminated for 90s each on the front and back sides. Demoulding, dialyzing with absolute ethyl alcohol and deionized water for 7 days, pre-freezing at-20 ℃, and freeze-drying to finally prepare the GelMA macroporous hydrogel scaffold for qualitative analysis of element content.

And (3) quenching and forging the freeze-dried bracket by using liquid nitrogen, then breaking the freeze-dried bracket, fixing the freeze-dried bracket on an electric microscope table by using conductive adhesive, spraying gold for 60s, carrying out EDS (electron-beam spectroscopy) test by using a scanning electron microscope, and qualitatively analyzing the element content of the GelMA macroporous hydrogel bracket. The overall scan results of the EDS spectra are shown in fig. 5a, and the local scan results are shown in fig. 5 b. And selecting a certain hole for scanning, wherein the content of Mg element in a circular area marked in the figure is 1.14 wt% and is higher than that in the whole visual field range.

Example 5 (Mg of GelMA macroporous hydrogel scaffold²⁺In vitro Release quantitative characterization

The freeze-dried hydrogel scaffolds of each group were weighed, and the scaffolds were placed in 24-well plates according to the concentration of 10mg of the scaffold immersed in 1ml of PBS (pH 7.4), respectively, and the corresponding amount of PBS solution was added for immersion. The magnesium ion release profile was obtained for each group by incubation in a 37 ℃ constant temperature shaker for 14 days. The shaker speed was 100 rpm. At each fixed time point, the PBS solution was removed and the release of magnesium ions was detected by inductively coupled plasma emission spectroscopy (ICP-OES, Perkin Elmer, Optima 2100DV, USA). The PBS solution was replaced with fresh one day. Mg of GelMA macroporous hydrogel scaffold²⁺In vitro release profiles are shown in fig. 6a and 6 b.

FIG. 6a shows the daily amount of released Mg element, Mg added by MgO²⁺The release of (1) is a burst release process, the content is high in the first two days, the content is reduced at the beginning of the third day, and after 7 days, the release amount tends to be flat and the concentration is low. The release amount in the first 5 days is 20-500 ppm. FIG. 6b shows the cumulative amount of Mg released within 10 days, and after 7 days, the release tended to be gentle.

Example 6 (characterization of internal morphology of GelMA macroporous hydrogel scaffolds with different MgO concentrations)

And (3) quenching and forging the freeze-dried hydrogel supports of each group by using liquid nitrogen, then breaking the freeze-dried hydrogel supports, fixing the freeze-dried hydrogel supports on an electric microscope table by using conductive adhesive, spraying gold for 60s, and observing the sections of the hydrogel supports of each group by using a scanning electron microscope so as to observe the internal appearance and aperture rule of the hydrogel supports. SEM photographs of GelMA macroporous hydrogel scaffolds with different MgO concentrations are shown in fig. 7. The hydrogel has no uniform pore structure, and the internal appearance of the hydrogel is shriveled and collapsed seriously due to the same treatment method because of the weak mechanical strength, and the pore walls are scaly.

0.3 wt% MgO group: has a uniform macroporous structure, and the aperture is about 180 mu m.

0.5 wt% MgO group: has a uniform macroporous structure, and the aperture is about 110 mu m.

0.7 wt% MgO group: the uniformity of the pores is poor, and the pore diameter is about 90 μm.

The aperture accords with the theory that the lower the MgO content is, the larger the aperture is, and the pore structure is more uniform.

Example 7 (cell compatibility test of GelMA macroporous hydrogel scaffolds with different MgO concentrations)

A hydrogel scaffold prepared from a PDMS mold with the thickness of 2mm and the diameter of 8mm is planted with mBMSCs of the 9 th generation after dialysis, freeze-drying and sterilization, and the planting density is 5 ten thousand cells/scaffold. The liquid was changed every two days.

The scaffolds cultured for 1,3 and 7 days are subjected to CCK-8 detection, and the absorbance of CCK-8 working solution is measured by a microplate reader to quantitatively analyze the proliferation condition of cells on the scaffolds. The proliferation of the cells at 1,3,7 days is shown in FIG. 8.

As can be seen from FIG. 8, only 0.5 wt% of the groups proliferated slightly by the time of culture to day 3, and none of the first three groups proliferated. By the time of culture to

day

7, 0 wt% of the nonporoous hydrogel group remained nonproliferating, 0.1 wt%, 0.3 wt% of the group slightly proliferated, and 0.5 wt% of the group significantly proliferated. It is shown that when the concentration of MgO is lower, compared with the nonporous gel, the MgO gel has the structural advantage of macropores and can promote cell proliferation, but the effect is not obvious; at a higher MgO concentration, the pore diameter is reduced, but the effect of Mg on promoting cell proliferation is more prominent.

The scaffolds cultured up to day 7 were subjected to Live/Dead staining, and the distribution of cells on the scaffolds and the state of the cells were observed using a confocal laser microscope. On day 7, a confocal laser photograph of Live/Dead staining is shown in FIG. 9.

The hydrogel scaffold with the macroporous structure has the advantages of better proliferation condition of cells on the scaffold, more cell quantity, better cell state and spreading and attaching state. Cells can penetrate deeper into the gel as they grow, rather than just growing on the surface of the scaffold.

The above-mentioned embodiments are merely preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, so that the changes in the shape and principle of the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for preparing GelMA macroporous hydrogel by a Pickering emulsion method is characterized in that firstly, methacrylic anhydride is used for carrying out graft modification on a gelatin molecular chain to obtain methacrylated gelatin, namely GelMA, double bonds on the gelatin undergo a thiol-ene click reaction with sulfydryl on PEG-4SH of four arms under the irradiation of UV light under the catalysis of a photoinitiator I2959 to quickly form hydrogel, an oil phase solution consisting of MgO nano particles and dodecane is added into a gel precursor solution, namely a water phase, a homogenizer is stirred at a high speed to prepare stable and uniform Pickering emulsion, then the gel is irradiated by the UV light to form gel, and dodecane oil drops serving as templates are washed off, so that the GelMA macroporous hydrogel taking the MgO nano particles as a Pickering emulsion stabilizer is obtained.

2. The method for preparing the GelMA macroporous hydrogel by the Pickering emulsion method according to claim 1, which is characterized by comprising the following steps:

1) dissolving GelMA and PEG-4SH in 1 XPBS buffer solution to obtain an aqueous phase solution;

3. The method for preparing GelMA macroporous hydrogel by Pickering emulsion method according to claim 2, wherein the method comprises the following steps: in the step 1), the substitution degree of GelMA is more than 70%, and the molecular weight of PEG is 2-20 kDa.

4. The method for preparing GelMA macroporous hydrogel by Pickering emulsion method according to claim 2, wherein the method comprises the following steps: in the step 1), the mass ratio of GelMA to PEG-4SH is 1: 0.5-1.

5. The method for preparing GelMA macroporous hydrogel by Pickering emulsion method according to claim 2, wherein the method comprises the following steps: in the step 2), the particle size of the MgO nano-particles is 20-100 nm.

6. The method for preparing GelMA macroporous hydrogel by Pickering emulsion method according to claim 2, wherein the method comprises the following steps: in the step 2), the ultrasonic time is 10-60 min.

7. The method for preparing GelMA macroporous hydrogel by Pickering emulsion method according to claim 2, wherein the method comprises the following steps: in the step 3), the addition amount of the I2959 photoinitiator is 0.01-0.1 wt%.

8. The method for preparing GelMA macroporous hydrogel by Pickering emulsion method according to claim 2, wherein the method comprises the following steps: in the step 3), the high-speed stirring speed of the homogenizer is 1-2 ten thousand revolutions per minute, and the stirring time is 30-90 s.

9. The method for preparing GelMA macroporous hydrogel by Pickering emulsion method according to claim 2, wherein the method comprises the following steps: in the step 4), the thickness of the PDMS mold is 2-5 mm, the aperture is 5-10 mm, and the UV illumination time is 30-90 s for each of the front side and the back side of the mold.