Janus hydrogel as well as preparation method and application thereof

Abstract

The invention discloses Janus hydrogel as well as a preparation method and application thereof, the Janus hydrogel comprises a gelatin base material loaded with bioactive molecules, and one surface of the base material is coated with a serine and / or derivative layer thereof. The gelatin base material containing active molecules in the Janus hydrogel is an anti-biological pollution and anti-inflammatory functional surface, adhesion, growth and ingrowth of fiber cells on the material can be effectively inhibited, bacterial infection and inflammatory reaction are reduced, and the side coated with the serine or serine derivative layer promotes growth of osteogenesis related cells, so that the effect of promoting osteogenesis is achieved. Comprising homing, adhesion and growth of bone marrow mesenchymal stem cells, differentiation of cells before osteogenesis, proliferation of osteoblasts and the like, the osteogenesis process is accelerated, and finally new bones are formed at bone defects.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a Janus hydrogel and a preparation method and application thereof. Background Art

[0002] Guided bone regeneration technology is a surgical method that places a physical barrier at the bone defect to isolate the connective tissue, thereby establishing a specific favorable environment to promote bone tissue regeneration. Guided bone regeneration membranes have been shown to play an important role in promoting bone tissue repair, expanding the alveolar ridge, promoting dental implant bone integration and periodontal tissue regeneration, and have been widely used in animal experiments and clinical practice. Tissue isolation and maintenance of regeneration space are two important features of guided bone regeneration membranes. Researchers have developed a variety of membrane materials, including synthetic polymers, natural polymers, metal materials and inorganic compounds. Among them, according to the biodegradability of the materials, they are divided into non-degradable materials represented by polytetrafluoroethylene (ePTFE), and degradable materials represented by previous collagen to today's polylactic acid (PLA). Non-degradable materials and degradable materials have their own advantages and disadvantages in clinical use as guided periosteum. Among them, the advantages of non-degradable materials are mainly that the membrane material is stable, has strong mechanical strength and regeneration space maintenance ability, is widely used, has a long history, and ensures reliable efficacy; the disadvantages are that the membrane material is hard, difficult to cut, shape, and fix, and the operation is time-consuming; long-term retention is prone to bacterial infection, requiring secondary surgery, and is expensive. On the premise that the membrane material can provide good tissue separation and space maintenance, the biggest advantage of degradable materials is that they do not require secondary surgery to remove. In addition, the fixation operation is easy, the physicochemical properties are easy to regulate, the biological activity accelerates bone formation and bone integration, and has antibacterial functions; its disadvantages are also obvious, which are also the problems faced by almost all degradable biomaterials, such as the controllability of the degradation rate and the retention of degradation products, which are very likely to lead to inflammatory reactions and foreign body reactions. With the development of modern medicine and the spread of the concept of regenerative medicine, people currently tend to choose degradable guided bone regeneration membranes in practical applications.

[0003] Although degradable guided bone regeneration membranes have excellent barrier effects, they often lack sufficient space maintenance capabilities, causing membrane collapse and leading to poor bone regeneration. For example, electrospinning membrane materials, inorganic powder pressed membranes or hydrophilic polymer membranes, etc., collapse in the body due to stress, hydrolysis or degradation, and even structural damage. Therefore, researchers use guided bone regeneration membranes and bone substitute materials together to ensure the maintenance of bone regeneration space. Researchers studied whether guided bone regeneration membranes were used after bone repair materials were implanted in bone defects. The results showed that the combined use of guided bone regeneration membranes and bone substitute materials was better than using them alone. However, based on the barrier function of guided bone regeneration membranes, commercially available degradable guided bone regeneration membranes are designed to organize the inward growth of fibroblasts into the tissue defect site, which often limits the biological activity and regeneration potential of guided bone regeneration membrane materials. In recent years, the asymmetric structural characteristics of Janus materials have attracted attention in the application of guided bone regeneration membranes. Researchers usually focus on the pores or density on both sides of the degradable guided bone regeneration membrane materials to achieve the construction of an asymmetric structure. The small pores or dense side of the material can prevent the growth of fibroblasts, while the large pores or looseness on the other side is conducive to bone formation. However, this type of guided bone regeneration membrane material with asymmetric pore structure needs to be improved in terms of anti-infection and anti-inflammation. Summary of the invention

[0004] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the object of the present invention is to provide a Janus hydrogel and a preparation method and application thereof.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] The first aspect of the present invention provides a Janus hydrogel, comprising a gelatin substrate loaded with bioactive molecules, one side of the substrate being coated with a layer of serine and / or its derivatives.

[0007] In the present invention, the gelatin substrate containing active molecules in the Janus hydrogel is an anti-biological contamination and anti-inflammatory functional surface, which can effectively inhibit the adhesion, growth and ingrowth of fibroblasts on the material, reduce bacterial infection and inflammatory response, and the side coated with a layer of serine or a serine derivative promotes the growth of bone formation-related cells, including the homing, adhesion and growth of bone marrow mesenchymal stem cells, the differentiation of pre-osteoblastic cells and the proliferation of osteoblasts, thereby accelerating the process of bone formation and ultimately forming new bone at the bone defect.

[0008] In some embodiments of the present invention, in the gelatin matrix, the mass ratio of gelatin to bioactive molecules is 10-30:0.001-5.

[0009] In some embodiments of the present invention, the mass concentration of serine and / or its derivatives in the serine and / or its derivatives layer is 10% to 60%.

[0010] In some embodiments of the present invention, the thickness of the gelatin substrate is 0.5-5 mm; the thickness of the serine and / or its derivative layer is 0.05-0.2 mm, such as 0.05-0.1 mm, 0.05-0.08 mm.

[0011] In some embodiments of the present invention, the gelatin includes at least one of pure gelatin, methacrylated gelatin, thiolated gelatin, gelatin modified with dopamine and its derivatives, and gelatin modified with norbornene.

[0012] In some embodiments of the present invention, the bioactive molecule comprises at least one of a growth factor, a rigid nanoparticle, an antimicrobial agent, and a polyhydroxy compound.

[0013] In some embodiments of the present invention, the growth factor is at least one of bone morphological protein-2 (BMP-2), transforming growth factor-β (TGF-β), vascular endothelial growth factor, platelet-derived growth factor, deferoxamine, Kartogenin (KGN), VEGF, dexamethasone, eugenol, chondroitin sulfate, and interleukin.

[0014] In some embodiments of the present invention, the rigid nanoparticles include one or more of silicon dioxide nanoparticles, lithium magnesium silicate nanoparticles, tricalcium phosphate, nano calcium carbonate particles, hydroxyapatite nanoparticles, iron oxide magnetic nanoparticles, barium titanate nanoparticles, graphene nanosheets, carbon nanotubes, nano copper oxide powder, bioglass nanoparticles, black phosphorus nanosheets, silk fibroin nanoparticles, polylactic acid nanoparticles, polyethylene nanoparticles, and polystyrene nanoparticles; the size of the rigid nanoparticles is 10nm to 50μm.

[0015] In some embodiments of the present invention, the antimicrobial agent comprises an antibiotic and / or polylysine.

[0016] In some embodiments of the present invention, the polyhydroxy compound is selected from at least one of dopamine, tannic acid, catechol or its derivatives, and mussel protein.

[0017] In some embodiments of the present invention, the serine and / or its derivatives include at least one of serine, homoserine, serineamide, and (meth)acrylserine.

[0018] In some embodiments of the present invention, the Janus hydrogel can be in any shape and can be adaptively tailored according to actual application needs. Common shapes include block, film, and micro hydrogel.

[0019] The second aspect of the present invention provides a method for preparing the Janus hydrogel, comprising the following steps:

[0020] The gelatin substrate loaded with bioactive molecules is immersed on one side in an aqueous solution of serine and / or its derivatives, and solidified to obtain the Janus hydrogel.

[0021] In some embodiments of the present invention, the immersion times are at least once, such as 1 to 10 times, 2 to 8 times, 3 to 6 times, etc., so that one side of the gelatin substrate is fully immersed in the aqueous solution of serine and / or its derivatives, thereby obtaining a hydrophilic coating.

[0022] In some embodiments of the present invention, the mass volume concentration of serine and / or its derivatives in the aqueous solution of serine and / or its derivatives is 10-60%.

[0023] In some embodiments of the present invention, the curing treatment includes any one of a carbodiimide salt method, a formaldehyde (glutaraldehyde) method, an ultraviolet light crosslinking technique, and a radiation crosslinking technique.

[0024] In some embodiments of the present invention, the curing process is performed by ultraviolet light cross-linking technology, including a light intensity of 15 to 50 mW / mm 2 Irradiate under ultraviolet light for 1 to 5 minutes.

[0025] In some embodiments of the present invention, the gelatin substrate loaded with bioactive molecules is prepared by any one of freezing technique, casting method or light curing technique.

[0026] In some embodiments of the present invention, the gelatin substrate loaded with bioactive molecules is prepared by cross-linking gelatin, bioactive molecules and an initiator in an aqueous solution.

[0027] In some embodiments of the present invention, in the reaction system, the mass volume concentration of gelatin is 10% to 30%; the mass volume concentration of the bioactive molecule is 0.01% to 5%; and the mass volume concentration of the initiator is 0.02% to 1%.

[0028] In some embodiments of the present invention, the initiator includes lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP).

[0029] The third aspect of the present invention provides the use of the Janus hydrogel in preparing guided bone regeneration materials.

[0030] In some embodiments of the present invention, the guided bone regeneration material includes at least one of a bone tissue repair material and a tooth repair material.

[0031] In some embodiments of the present invention, the bone tissue repair material includes a bone / cartilage integrated repair material.

[0032] The beneficial effects of the present invention are:

[0033] The Janus hydrogel of the present invention presents two sides with different characteristics. One side presents excellent anti-biological contamination and anti-inflammatory functions, effectively inhibiting the adhesion, growth and ingrowth of fibroblasts on the material, reducing bacterial infection and inflammatory response, while the other side of the Janus hydrogel membrane accelerates the process of bone formation by promoting the growth of bone formation-related cells, including the homing, adhesion and growth of bone marrow mesenchymal stem cells, the differentiation of pre-osteoblastic cells and the proliferation of osteoblasts, and ultimately forms new bone at the bone defect.

[0034] The preparation method of the Janus hydrogel of the invention is simple and easy to industrialize. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The anti-protein adsorption effect of the hydrogels of Example 1 and Comparative Examples 1 and 2 of the present invention.

[0036] Figure 2 The antibacterial effects of the hydrogels of Example 1 and Comparative Examples 1 and 2 of the present invention are shown.

[0037] Figure 3 The cell attachment behavior on the hydrogel surface of Example 1 of the present invention and Comparative Examples 1 and 2. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0039] Example 1

[0040] This example prepares a Janus hydrogel, and the specific process is as follows:

[0041] Weigh 2 g of methacrylated gelatin and 0.1 g of hydroxyapatite in 10 mL of 0.1% LAP solution, stir and dissolve at 60°C to obtain a prepolymer solution, stir for a period of time to make it fully dissolved and uniform; then transfer the prepared prepolymer solution to a rectangular template and irradiate it with ultraviolet light for 2 minutes at a light intensity of 30 mW / mm 2, curing and cross-linking to prepare a hydrogel substrate, immersing one side of the prepared hydrogel substrate sheet in a LAP solution containing 40% acrylated serinamide, taking it out after 5 minutes, irradiating it with ultraviolet light for 1 minute, and repeating it 3 times. Finally, the Janus hydrogel membrane material for guided bone regeneration was prepared, denoted as Gel-1, of which the untreated substrate surface was denoted as Gel-1@A, and the treated substrate surface was denoted as Gel-1@B.

[0042] Example 2

[0043] This example prepares a Janus hydrogel, and the specific process is as follows:

[0044] Weigh 2 g of methacrylated gelatin and 0.1 g of laponite in 10 mL of 0.1% LAP solution, stir and dissolve at 60 °C to obtain a prepolymer solution, stir for a period of time to make it fully dissolved and uniform; then transfer the prepared prepolymer solution to a rectangular template and irradiate it with ultraviolet light for 2 minutes at a light intensity of 30 mW / mm 2 The hydrogel substrate was prepared by curing and cross-linking, and one side of the prepared hydrogel substrate sheet was immersed in a LAP solution containing 40% thiolated serine, taken out after 5 minutes, and irradiated with ultraviolet light for 1 minute, and repeated 3 times. Finally, the Janus hydrogel membrane material for guided bone regeneration was prepared.

[0045] Example 3

[0046] This example prepares a Janus hydrogel, and the specific process is as follows:

[0047] 2 g of methacrylated gelatin and thiol-modified gelatin and 0.1 g of hydroxyapatite were weighed and dissolved in 10 mL of 0.1% LAP solution at 60 °C to obtain a prepolymer solution, which was stirred for a period of time to fully dissolve. The prepared prepolymer solution was then transferred to a rectangular template and irradiated with ultraviolet light for 2 minutes at a light intensity of 30 mW / mm 2 The hydrogel substrate was prepared by curing and cross-linking, and one side of the prepared hydrogel substrate sheet was immersed in a LAP solution containing 40% propylene serine, taken out after 5 minutes, and irradiated with ultraviolet light for 1 minute, and repeated 3 times. Finally, the Janus hydrogel membrane material for guided bone regeneration was prepared.

[0048] Comparative Example 1

[0049] This comparative example prepared a hydrogel, and the specific process was as follows:

[0050] Weigh 2 g of methacrylated gelatin and 0.1 g of hydroxyapatite in 10 mL of 0.1% LAP solution, stir and dissolve at 60°C to obtain a prepolymer solution, stir for a period of time to make it fully dissolved and uniform; then transfer the prepared prepolymer solution to a rectangular template and irradiate it with ultraviolet light for 2 minutes at a light intensity of 30 mW / mm 2 , and a hydrogel was prepared by curing and cross-linking, which was recorded as Gel-2.

[0051] Comparative Example 2

[0052] This comparative example prepared a hydrogel, and the specific process was as follows:

[0053] Weigh 4 g of acrylated serine amide in 10 mL of 0.1% LAP solution, stir and dissolve at room temperature to prepare a 40% acrylated serine amide solution, then transfer the prepared solution to a rectangular template and irradiate with UV light for 5 minutes at a light intensity of 30 mW / mm 2 , and a hydrogel was prepared by curing and cross-linking, which was recorded as Gel-3.

[0054] Test Example 1

[0055] This test example tests the anti-protein adsorption effect of the hydrogel, and the specific process is as follows:

[0056] Prepared 10mm diameter disc-shaped Gel-1 (divided into two groups: Gel-1@A facing up and Gel-1@B facing up), Gel-2 and Gel-3, put them into a 24-well plate, and then add 1mL of bovine serum albumin phosphate buffer solution. After incubation at 37°C for 4 hours, wash with phosphate buffer solution 3 times, and test the protein adsorption amount with a UV spectrophotometer. The anti-protein adsorption effect of the hydrogel was evaluated according to the absorbance of the solution. The results are as follows: Figure 1 shown.

[0057] like Figure 1 As shown, in the Janus hydrogel Gel-1, the protein adsorption amount of Gel-1@A surface was 98.54±2.42g, and the protein adsorption amount of Gel-1@A surface was 6.32±3.91g. The protein adsorption amount of Gel-1@A surface was significantly higher than that of Gel-1@B surface; the protein adsorption of hydrogel Gel-2 was 96.34±1.76g, and the protein adsorption amount of hydrogel Gel-3 was 6.51±3.02g. The protein adsorption amount of hydrogel Gel-2 was significantly higher than that of hydrogel Gel-3. The above results indicate that Gel-3 and Gel-1@B surface have good anti-protein nonspecific adsorption capabilities.

[0058] Test Example 2

[0059] This test example tests the antibacterial effect of the hydrogel, and the specific process is as follows:

[0060] Prepare 10 mm diameter disc-shaped Gel-1 (divided into two groups, Gel-1@A facing up and Gel-1@B facing up), Gel-2 and Gel-3, put them into a 24-well plate, and then add Escherichia coli solution at a quantity of 10,000 per well. After incubation at 37°C for 24 hours, wash three times with phosphate buffer solution, fix the samples with 4% paraformaldehyde, wash with phosphate buffer solution, wash and dehydrate with gradient ethanol solution, and observe the bacteria on the sample surface with a scanning electron microscope after drying.

[0061] like Figure 2 As shown in the figure, the number of Escherichia coli colonies on the Gel-1@A surface of Janus hydrogel Gel-1 is higher than that on the Gel-1@B surface. By using Image J software analysis, it is known that the area of ​​Escherichia coli on the Gel-1@A surface accounts for 66.35±3.45%, and the area of ​​Escherichia coli on the Gel-1@B surface accounts for 3.28±0.34%. The number of Escherichia coli colonies in hydrogel Gel-2 is significantly higher than that in hydrogel Gel-3. By using Image J software analysis, it is known that the area of ​​Escherichia coli on the Gel-1@A surface accounts for 70.09±1.75%, and the area of ​​Escherichia coli on the Gel-1@B surface accounts for 3.78±0.74%. The above results indicate that Gel-3 and Gel-1@B surfaces have good anti-bacterial adhesion effects, thereby resisting bacterial colonization on the gel surface and reducing the possibility of bacterial infection.

[0062] Test Example 3

[0063] This test example tests the cell attachment behavior on the hydrogel surface. The specific process is as follows:

[0064] Prepare 10 mm diameter disc-shaped Gel-1 (divided into two groups: Gel-1@A facing up and Gel-1@B facing up), Gel-2 and Gel-3, put them into a 24-well plate, and then add NIH 3T3 fibroblasts at a number of 10,000 per well. After culturing in a cell culture incubator for 24 hours, the cell attachment behavior on the material surface was observed under a microscope.

[0065] like Figure 3As shown in the figure, the number of NIH 3T3 fibroblasts on the Gel-1@A surface of Janus hydrogel Gel-1 was significantly higher than that on the Gel-1@B surface. The cells on the Gel-1@A surface could adhere to the gel surface with normal morphology and spindle shape, while the cells on the Gel-1@B surface were difficult to adhere to the gel surface and had a round morphology. Similarly, the number and morphology of NIH 3T3 fibroblasts on the hydrogel Gel-2 were better than those on the hydrogel Gel-3. By using Image J software analysis, it was found that the area of ​​NIH 3T3 fibroblasts on the Gel-1@A surface accounted for 95.27±3.63%, the area of ​​NIH 3T3 fibroblasts on the Gel-1@B surface accounted for 15.84±1.35%, the area of ​​NIH 3T3 fibroblasts on the Gel-2 surface accounted for 92.35±4.45%, and the area of ​​NIH The area of ​​3T3 fibroblasts accounted for 12.28±3.34%, indicating that Gel-1@A surface and Gel-2 can promote the attachment, spreading and growth of osteoblasts, osteocytes and other cells, while Gel-1@B surface and Gel-3 can effectively inhibit the attachment, growth and proliferation of fibroblasts on the hydrogel.

[0066] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A Janus hydrogel, characterized in that: The invention comprises a gelatin substrate loaded with bioactive molecules, one side of the substrate is coated with a layer of serine and / or its derivatives.

2. The Janus hydrogel according to claim 1, characterized in that: In the gelatin matrix, the mass ratio of gelatin to bioactive molecules is 10-30:0.001-5.

3. The Janus hydrogel according to claim 1, characterized in that: The mass concentration of serine and / or its derivatives in the serine and / or its derivatives layer is 10% to 60%.

4. The Janus hydrogel according to claim 1, characterized in that: The thickness of the gelatin substrate is 0.5-5 mm; the thickness of the serine and / or its derivative layer is 0.05-0.2 mm.

5. The Janus hydrogel according to claim 1, characterized in that: The bioactive molecules include at least one of growth factors, rigid nanoparticles, antimicrobial agents, and polyhydroxy compounds.

6. The Janus hydrogel according to claim 1, characterized in that: The serine and / or its derivatives include at least one of serine, homoserine, serineamide and (meth)acrylserine.

7. A method for preparing the Janus hydrogel according to any one of claims 1 to 6, characterized in that: The following steps are involved: The gelatin substrate loaded with bioactive molecules is immersed on one side in an aqueous solution of serine and / or its derivatives, and solidified to obtain the Janus hydrogel.

8. The method for preparing the Janus hydrogel according to claim 7, characterized in that: The dipping times is at least once.

9. Use of the Janus hydrogel according to any one of claims 1 to 6 in the preparation of guided bone regeneration materials.

10. The use according to claim 9, characterized in that: The guided bone regeneration material includes at least one of a bone tissue repair material and a tooth repair material.

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