阅读说明：本技术 一种poss增强水凝胶及其制备方法和用途 (POSS (polyhedral oligomeric silsesquioxane) reinforced hydrogel as well as preparation method and application thereof ) 是由 陈明姣 李瑾 张远豪 姜芳 张伟安 于 2020-05-21 设计创作，主要内容包括：本发明涉及生物医用水凝胶材料技术领域,特别是涉及一种POSS增强水凝胶及其制备方法和用途。本发明以透明质酸和POSS作为水凝胶的主体材料,基于透明质酸与POSS的共价交联及两种天然高分子的静电作用制备得到了POSS增强水凝胶,该水凝胶不仅结构稳定、热学性能和机械性能高,同时无毒、有良好的生物相容性。成凝胶方法简单、多样化,能更广泛的应用于组织工程。(The invention relates to the technical field of biomedical hydrogel materials, in particular to a POSS (polyhedral oligomeric silsesquioxane) reinforced hydrogel and a preparation method and application thereof. According to the invention, hyaluronic acid and POSS are used as main materials of the hydrogel, and the POSS reinforced hydrogel is prepared based on covalent crosslinking of hyaluronic acid and POSS and electrostatic action of two natural macromolecules. The gel forming method is simple and diversified, and can be widely applied to tissue engineering.)

1. A method for preparing a POSS reinforced hydrogel, comprising the steps of:

1) mixing hyaluronic acid and POSS;

2) adding a photoinitiator;

3) irradiating the system in the step 2) under ultraviolet light to obtain the POSS reinforced hydrogel.

2. The method of claim 1, wherein step 1) further comprises gelatin.

3. The method according to claim 2, wherein the hyaluronic acid is a modified hyaluronic acid, and/or the POSS is a modified POSS, and/or the gelatin is a modified gelatin.

4. The method according to claim 1, wherein the hyaluronic acid is a hyaluronic acid modified with a hydrophobic segment or a hydrophobic group.

5. The method of claim 2, further comprising one or more of the following features:

1) the hyaluronic acid is selected from HAMA, and the HAMA is hyaluronic acid modified by methacrylate;

2) the POSS is selected from OMAPOSS;

3) the gelatin is selected from methacrylic gelatin or aminated gelatin;

4) the photoinitiator is selected from LAP.

6. The method of claim 2, further comprising one or more of the following features:

1) the solvent in the step 1) is dimethyl sulfoxide;

2) the ultraviolet irradiation time in the step 3) is 10-30 minutes;

3) based on the mass of the solvent, the concentration of the hyaluronic acid is 1-3 wt%;

4) based on the mass of the solvent, the concentration of POSS is 1-5 wt%;

5) based on the mass of the solvent, the concentration of the gelatin is 1 wt% -3 wt%.

7. The POSS reinforced hydrogel is characterized by being obtained by carrying out a crosslinking reaction on hyaluronic acid and POSS.

8. A POSS reinforced hydrogel according to claim 7, wherein said POSS reinforced hydrogel is obtained by the production method as set forth in any one of claims 1 to 6.

9. Use of a POSS-reinforced hydrogel as claimed in claim 7 or 8 in the manufacture of a bone repair product, a cell proliferation or differentiation product.

10. Use of a POSS-enhanced hydrogel as claimed in claim 9 for the preparation of a product for stimulating the expression of osteogenic differentiation genes in cells.

Technical Field

The invention relates to the technical field of biomedical hydrogel materials, in particular to a POSS (polyhedral oligomeric silsesquioxane) reinforced hydrogel and a preparation method and application thereof.

Background

The hydrogel is a material with a three-dimensional cross-linked network structure which has high water content and is relatively soft, and has wide application prospect in tissue engineering. However, the non-uniformity of the hydrogel structure and the lack of energy dissipation limit the application of the hydrogel to tissues with high requirements on mechanical strength, such as cartilage and tendon tissue. Although the improvement of the mechanical strength of the hydrogel by increasing the degree of crosslinking of a single substance is successful, the toughness of the obtained material is poor, and the further application of the material is hindered, so that the improvement of the mechanical strength and the improvement of the toughness are important problems for widening the application of the hydrogel.

POSS (cage polysilsesquioxane) is an inorganic compound with a nano cage structure alternately connected by silicon-oxygen bonds; the diameter of POSS is about 1.5nm, and one or more functional groups can be connected at the tail end of POSS, so that the use of POSS is greatly enriched. Despite the numerous advantages of POSS, POSS-reinforced hydrogels generally are based on oil-soluble synthetic polymers due to the hydrophobic silica structure of POSS. However, most synthetic macromolecules have poor biological properties and lack certain cell regulation mechanisms. While another source of hydrogels, natural polymers, play an important role in cell, tissue compatibility and bio-induction, unfortunately, water-soluble natural polymers are difficult to react homogeneously with hydrophobic POSS in the same solvent.

Hyaluronic Acid (HA) is a natural mucopolysaccharide composed of a repeat of D-glucuronic acid and N-acetyl-D-glucosamine, is also a major component of extracellular matrix (ECM), is widely present in tissues such as skin and cartilage, can specifically bind to various cell receptors to regulate the characteristics of cells such as growth, separation, migration, etc., can be decomposed into amino acids absorbable by hyaluronidase in the body, and HAs good biocompatibility and biodegradability. Gelatin is a degradation product of collagen, has abundant polypeptides, retains the characteristics of collagen, and has good biocompatibility and biodegradability. The related literature also reports that gelatin is less antigenic than collagen. However, the mechanical properties of hyaluronic acid gel and gelatin gel are poor, and the application of hyaluronic acid gel and gelatin gel in tissue engineering is limited to a certain extent.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide a POSS reinforced hydrogel, a method of making the same, and uses thereof, which solve the problems of the prior art.

To achieve the above and other related objects, the present invention provides a method for preparing POSS reinforced hydrogels, comprising the steps of:

1) mixing hyaluronic acid and POSS;

2) adding a photoinitiator;

3) irradiating the system in the step 2) under ultraviolet light to obtain the POSS reinforced hydrogel.

Specifically, the hyaluronic acid is modified hyaluronic acid.

In particular, the modified hyaluronic acid incorporates a hydrophobic segment or group.

More preferably, the hyaluronic acid is methacrylic acid modified hyaluronic acid, abbreviated as HAMA. The mass fraction of the hyaluronic acid is 1 wt% -3 wt% based on the mass of the solvent.

Specifically, the POSS is modified POSS.

Preferably, the modified POSS is omapos (octamethacrylate-based oligomeric silsesquioxane).

During preparation, the POSS accounts for 1-5 wt% of the mass of the solvent.

Preferably, gelatin is also added when preparing POSS reinforced hydrogels.

The gelatin is modified gelatin.

Preferably, the gelatin is methacrylic gelatin or aminated gelatin.

The preparation method takes the mass of the solvent as the reference, and the mass fraction of the gelatin is 1 to 3 weight percent

Specifically, the photoinitiator is LAP.

In the preparation step 2), the mass fraction of LAP is 0.07-0.13 wt% based on the mass of the solvent.

Specifically, in the step 3) of the preparation method, ultraviolet light is used for irradiating for 10-30 minutes.

Specifically, the solvent used in the preparation method is dimethyl sulfoxide.

The invention also provides the POSS reinforced hydrogel prepared by the preparation method.

The invention finally provides the use of POSS-enhanced hydrogels for the preparation of cell proliferation or differentiation products.

The POSS enhanced hydrogel can stimulate the expression of osteogenic differentiation genes of cells.

As mentioned above, the POSS reinforced hydrogel and the preparation method and the application thereof have the following beneficial effects:

1) the structure is stable, and the thermal property and the mechanical property are high;

2) no toxicity and good biocompatibility;

3) the gel forming mode is simple and diversified, and can be widely applied to tissue engineering such as bone repair and the like.

Drawings

FIG. 1 shows a schematic representation of the HA-POSS-Gel hydrogel composition.

FIG. 2 shows NMR spectra of Hyaluronic Acid (HA) and double bond modified hyaluronic acid (HAMA).

FIG. 3 shows the hydrogen nuclear magnetic resonance spectrum of LAP.

FIG. 4(a) shows a solution of d-HAMA mixed with Gel-ADH, and FIG. 4(b) shows a solution of d-HAMA mixed with Gel-ADH.

FIG. 5 shows FTIR spectra of HA-POSS-Gel hydrogel.

FIG. 6 is an appearance chart of the hydrogel prepared in example 4, wherein (a) to (e) are HA-POSS-Gel hydrogels with final concentrations of 0%, 1%, 2%, 3%, 4% to which OMAPOSS was added, respectively, f is a hydrogel obtained by adding only dHAMA, and g is an HA-POSS hydrogel obtained by adding dHAMA and OMAPOSS, wherein the final concentrations of dHAMA and OMAPOSS were 3%.

FIG. 7 shows SEM images of HA-POSS-Gel hydrogels, wherein (a) - (e) are the internal morphologies of the hydrogels with the addition of OMAPOSS at final concentrations of 0%, 1%, 2%, 3%, 4%, respectively, and the scale is 200 μm.

FIG. 8 shows the data for the compression performance test of HA-POSS-Gel hydrogels: (a) the stress-strain curve of the HA-Gel hydrogel, (b) the compressive modulus of the HA-Gel hydrogel, (c) the stress-strain curve of the HA-POSS-Gel hydrogel, and (d) the compressive modulus of the HA-POSS-Gel hydrogel.

FIG. 9 shows the stress curves for 3 consecutive loading-unloading of HA-POSS-Gel hydrogels, prepared at 1%, 2%, 3% and 4% final concentrations of OMAPOSS (a), (b), (c) and (d), respectively.

FIG. 10 shows the dissipation energy of the HA-POSS-Gel hydrogel obtained from the first load-unload stress curve of FIG. 9.

FIG. 11 shows the rotational rheological properties of the HA-POSS-Gel hydrogel at 0.1% strain, 1-100rad/s, with G' and G "representing the storage and loss moduli of the hydrogel, respectively.

FIG. 12 shows the swelling behavior of HA-POSS-Gel hydrogels.

Figure 13 shows the degradation performance of the hydrogel in hyaluronidase.

FIG. 14 shows the dead and live fluorescence staining of MSCs after incubation with HA-POSS-Gel hydrogel, where (a) - (e) are hydrogels with final concentrations of OMAPOSS of 0%, 1%, 2%, 3%, 4%, respectively, where green is live cells, red is dead cells, and the scale is 200 μm.

FIG. 15-1 shows the proliferation of cells on HA-POSS-Gel hydrogels, where (a) - (e) are hydrogels with final concentrations of OMAPOSS of 0%, 1%, 2%, 3%, 4%, respectively.

FIG. 15-2 shows the proliferation of cells on HA-POSS and HA hydrogels.

FIG. 16-1 shows the osteogenic differentiation of cells on HA-POSS-Gel hydrogels, where (a) - (e) are hydrogels with final concentrations of OMAPOSS of 0%, 1%, 2%, 3%, 4%, respectively.

FIG. 16-2 shows osteogenic differentiation of cells on HA-POSS and HA hydrogels.

In the above-described drawings, unless otherwise specified,

error bars indicate Standard Deviation (SD)

"+" represents 0.01 ≦ P <0.05

"x" represents P < 0.01.

Detailed Description

Through intensive research, the inventors of the present invention invented a preparation method of POSS reinforced hydrogel, comprising the steps of:

1) mixing hyaluronic acid and POSS;

2) adding a photoinitiator;

3) irradiating the system in the step 2) under ultraviolet light to obtain the POSS reinforced hydrogel.

The POSS reinforced hydrogel is a nano composite hydrogel material formed by covalent crosslinking of POSS serving as a performance-reinforced nano particle and a matrix.

The matrix comprises hyaluronic acid, and when two natural polymers, namely the matrix and POSS, coexist, an electrostatic effect exists between the two natural polymers, so that hydrogel formation is facilitated.

In particular, the hyaluronic acid is a modified hyaluronic acid, i.e. a modified hyaluronic acid.

Specifically, the modified hyaluronic acid is introduced with a hydrophobic segment or group, such as C10-C20 alkyl, alkyl containing aryl, ester, ether, amine, amide and other groups, or other double bond-containing alkyl.

In one embodiment, the modified hyaluronic acid has been grafted with a substantial amount of alkyl groups containing ester groups, with a grafting yield of at least 50%.

In a preferred embodiment, the hyaluronic acid is methacrylic acid modified hyaluronic acid, abbreviated as HAMA, which can be a commercially available reagent or can be synthesized in the laboratory. The mass fraction of the hyaluronic acid is 1 wt% -3 wt% based on the mass of the solvent.

The modified hyaluronic acid can be dissolved in DMSO, so that the water-soluble hyaluronic acid and the hydrophobic POSS can uniformly react in the same solvent.

Specifically, POSS (polyhedral oligomeric silsesquioxane) is a novel organic-inorganic nano hybrid material with a stable structure and a nano size, and can be uniformly distributed in various base materials as a cross-linking agent.

Specifically, the POSS is modified POSS.

In one embodiment, the modified POSS is OMAPOSS (octamethacrylate-based oligomeric silsesquioxane) having the following structural formula:

OMAPOSS is a POSS with 8 double bond active groups at the end, and is used as a cross-linking agent to improve the overall mechanical properties of the hydrogel.

During preparation, the POSS accounts for 1-5 wt% of the mass of the solvent.

In one embodiment, gelatin is also added during the preparation of the POSS reinforced hydrogel, and the hydrogel prepared by adding the gelatin has better mechanical property, good biocompatibility and slower degradation speed, and is more favorable for the adhesion, the spreading and the differentiation of cells.

The gelatin is modified gelatin.

In one embodiment, the gelatin is a methacrylate gelatin having the formula:

in another embodiment, the gelatin is an aminated gelatin having a large number of amino groups obtained by reacting gelatin with adipic Acid Dihydrazide (ADH) and 1-hydroxybenzotriazole (HOBt).

The amino content detected on the aminated gelatin by an ultraviolet-visible spectrophotometry is 0.527 +/-0.008 mu mol/mg

The mass fraction of the gelatin is 1-3 wt% based on the mass of the dimethyl sulfoxide.

Specifically, the photoinitiator is phenyl-2, 4, 6-trimethylbenzoylphosphinic acid Lithium (LAP).

In the step 2) during the preparation, the LAP may be present in an amount of 0.07 to 0.13 wt%, for example, 0.1 wt% based on the mass of dimethyl sulfoxide.

In one embodiment, step 3) is performed after the solution in step 2) is completely clear.

Specifically, in the step 3) of the preparation method, the ultraviolet light is used for irradiating for 10-30 minutes, the specific irradiation time is different according to the power and wavelength of the ultraviolet lamp, and for example, the power can be 8mW/cm²For 10 minutes under a 365nm UV lamp.

And after the ultraviolet irradiation is finished, the POSS reinforced hydrogel is obtained.

Specifically, the solvent used in the preparation method is dimethyl sulfoxide.

The invention also provides the POSS reinforced hydrogel prepared by the preparation method. The POSS reinforced hydrogel has mechanical properties such as compression modulus, elongation at break, dissipation energy and the like which are superior to those of the POSS-free hydrogel, has swelling property, degradation property and internal microporous structure which are superior to those of the POSS-free hydrogel, particularly has more compact pore channels in the internal microporous structure, smaller pore structures among tube walls and a macroporous structure, and lays a foundation for the application of the POSS reinforced hydrogel in cells.

The invention also provides the use of POSS-enhanced hydrogels for the preparation of cell proliferation or differentiation products.

The common hyaluronic acid hydrogel is poor in mechanical property, lacks necessary adhesion units for cells, and the cells are not easy to adhere and grow.

The POSS reinforced hydrogel is applied to the preparation of products for stimulating the expression of osteogenic differentiation genes of cells.

The osteogenic differentiation gene comprises one or more of OPN, OSX, BSP and Runx 2.

In one embodiment, the cell is a mesenchymal stem cell.

Use of the POSS reinforced hydrogel in the preparation of a bone repair product.

The product necessarily includes a POSS reinforced hydrogel as an effective ingredient for the aforementioned effects.

Including but not limited to pharmaceuticals, agents, and the like.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.