CN111961227A

CN111961227A - Graphene quantum dot/hydrogel composite material, and preparation method and application thereof

Info

Publication number: CN111961227A
Application number: CN202010839771.5A
Authority: CN
Inventors: 沈龙祥; 耿弼江; 潘登余
Original assignee: Shanghai Sixth Peoples Hospital
Current assignee: Shanghai Sixth Peoples Hospital
Priority date: 2020-08-19
Filing date: 2020-08-19
Publication date: 2020-11-20
Anticipated expiration: 2040-08-19
Also published as: CN111961227B

Abstract

The invention discloses a graphene quantum dot/hydrogel composite material, which comprises a cross-linked network and graphene quantum dots adsorbed on the cross-linked network; the crosslinked network comprises a biocompatible macromolecule and a crosslinking agent; the cross-linking agent contains a carbon-carbon double bond and a carboxyl group. According to the invention, the GQDs with osteogenic differentiation inducing potential are loaded on the hydrogel to form a composite material, so that the defect that the hydrogel can not effectively promote osteogenic differentiation is overcome, the defect that the GQDs can not be condensed and formed, and can be singly applied to the body as a bone defect repair support is overcome, and a new possible support material is provided for promoting the healing of bone defect parts in the body as soon as possible.

Description

Graphene quantum dot/hydrogel composite material, and preparation method and application thereof

Technical Field

The invention relates to preparation of hydrogel loaded with Graphene Quantum Dots (GQDs) with osteogenic differentiation inducing potential and application of the hydrogel in bone tissue engineering. How to promote the healing of massive bone defects as soon as possible is always a research hotspot in the related field of orthopedics. The composite bone defect repairing scaffold material prepared by the invention has good osteogenic differentiation promoting effect, has good biocompatibility, is non-toxic, high in safety, simple in preparation process and low in cost, and provides a possible scaffold material for repairing bone defects clinically.

Background

The large area of bone defects presents a significant challenge to the repair of bone tissue. Autologous bone grafting is a widely applied method at present, has no self immune rejection reaction and has the functions of bone conduction and bone induction, but the method is limited by donor site complications and insufficient supply of transplanted bones. Metal fixation devices such as titanium cages, bone plates, etc. are commonly used in combination with implants to attach and stabilize bone fragments, but the metal material does not degrade in the body, and long-term retention in the body may cause discomfort and a series of complications. Bioceramics, bioactive glasses, and polymers with good biocompatibility are increasingly used in the repair of bone defects in vivo, and these materials are usually osteoconductive but usually not osteoinductive.

As a zero-dimensional carbon-based nano material, the GQDs inherits the unique physical, chemical and mechanical properties of the two-dimensional graphene, has excellent optical properties, excellent biocompatibility and adjustable surface functions, and has wide application prospects in biological imaging, biosensing, drug delivery, antibiosis and cancer treatment. In recent years, the application of GQDs in the biomedical field has been expanded to the fields of tissue engineering and regenerative medicine. However, GQDs cannot be coagulated by themselves, and thus, they are not effective when applied alone to the inside of living bodies.

The hydrogel is a three-dimensional reticular polymeric material formed by combining monomers and hydrophilic groups through a chemical or/and physical crosslinking method, and the hydrophilic structure of the hydrogel enables the hydrogel to have strong water absorption and water retention. The use of hydrogels in soft tissue engineering has been extensively studied, but the use in hard tissue regeneration has been rarely studied. The stem cells preserved in the three-dimensional structure of the hydrogel show higher proliferation and osteogenic differentiation capacities, because the hydrogel scaffold has excellent biocompatibility, degradability and structural controllability, so that the hydrogel scaffold can provide a good bioactive environment for cell proliferation and differentiation. However, hydrogels also have their own deficiencies: stem cells cannot be effectively promoted to differentiate toward osteoblasts.

Therefore, the invention improves the defect that the hydrogel can not effectively promote osteogenic differentiation by loading the GQDs with osteogenic differentiation inducing potential on the hydrogel, overcomes the defect that the GQDs can not be condensed and formed by the hydrogel and can be independently applied to the body as a bone defect repair bracket, thereby providing a new possible bracket material for promoting the healing of bone defect parts in the organism as soon as possible.

Disclosure of Invention

The invention aims to provide a novel in-vivo osteogenesis differentiation promoting and inducing reagent hydrogel composite material, a preparation method thereof and application thereof in promoting osteogenesis in vivo.

The graphene quantum dot/hydrogel composite material comprises a cross-linked network and graphene quantum dots adsorbed on the cross-linked network;

the crosslinked network comprises a biocompatible macromolecule and a crosslinking agent;

the cross-linking agent contains a carbon-carbon double bond and a carboxyl group.

As an embodiment, the biocompatible macromolecule is selected from at least one of gelatin, chitosan, hyaluronic acid, collagen;

the cross-linking agent is at least one selected from methacrylic acid, acrylic acid and crotonic acid.

As an embodiment, the crosslinked network and the graphene quantum dots are adsorbed by hydrogen bonds or electrostatic interaction.

As an embodiment, in the graphene quantum dot/hydrogel composite material, the following mass ratios of the components are satisfied:

biocompatible macromolecules: crosslinking agent = (0.5-1): (0.5-0.8);

cross-linking the network: graphene quantum dots = (300-: (0.1-0.3).

Preferably, in the graphene quantum dot/hydrogel composite material, the following components are satisfied:

biocompatible macromolecules: crosslinker = 10: 8;

cross-linking the network: graphene quantum dots = 300: 0.2.

as an embodiment, the graphene quantum dot/hydrogel composite material is a porous structure.

According to another aspect of the invention, the preparation method of the graphene quantum dot/hydrogel composite material is simple and convenient to operate, stable and controllable in reaction, and suitable for industrial production.

The preparation method of the hydrogel composite material comprises the following steps:

a) reacting a solution containing a biocompatible macromolecule and a cross-linking agent to obtain a first solution;

b) dialyzing the first solution to obtain a second solution;

c) lyophilizing the second solution to obtain a hydrogel precursor;

d) and reacting the solution containing the hydrogel composite material precursor and the graphene quantum dots in the presence of an initiator to obtain the graphene quantum dot/hydrogel composite material.

In one embodiment, in step a),

the reaction temperature is 30-50 ℃; the reaction time was 120-180 minutes.

Preferably, in step a), the temperature of the reaction is 40 ℃; the reaction time was 10 minutes.

In one embodiment, in step b),

the dialysis temperature is 40-50 ℃; the dialysis time is 3-5 days.

In one embodiment, in step c),

the initiator is selected from at least one of water-soluble initiator and photoinitiator;

the mass ratio of the hydrogel composite precursor to the initiator is (1-3): (0.005-0.015).

Preferably, the mass ratio of the hydrogel precursor to the initiator is 3: 0.01.

Preferably, the initiator is a photoinitiator;

the reaction conditions are as follows: and reacting for 5-15 minutes under ultraviolet light.

As a specific embodiment, the preparation method of the graphene quantum dot/hydrogel comprises the following steps:

1. preparing GQDs/hydrogel composite nanoparticles:

a) preheating 1 g of gelatin and 10mL of PBS at 50 ℃ for 20 minutes, and then adding 0.8 mL of methacrylic acid and stirring vigorously; preheating PBS for 10 minutes at 40 ℃, and adding 40 mL of PBS into the mixed solution to terminate the reaction; (ii) a

b) The solution is added at 40-50 deg.C^oDialyzing for 3 days under the condition of C;

c) freeze-drying the solution in (c) by using a freeze dryer.

d) 0.3 g of the lyophilized solid in the form of a foam was taken, and 2 mL of PBS and 200 mL (1 mg mL) were added^-1) And finally adding 0.01 g of photosensitizer, and continuously irradiating for 10 minutes under an ultraviolet lamp until the hydrogel is solidified.

Cytotoxicity of GQDs/hydrogel composite nanoparticles on bone marrow human mesenchymal stem cells (hMSCs):

cell viability of hMSCs was determined using MTT. Cell viability is expressed as the ratio of the total number of viable cells to the total number of cells. Firstly, preparing leachate of hydrogel/GQDs composite material, immersing blank hydrogel or hydrogel/graphene quantum composite material into hMSCs culture medium, shaking, and placing in an incubator at 37 ℃ for 24 hours. Filtering, sterilizing, and storing at 4 deg.C. Next, hMSCs were seeded in 96-well plates and placed in CO₂The culture box was incubated for 24 hours, and the basal medium for stem cells was replaced with 80. mu.L of basal medium and 20. mu.L of leachate. After 24/48 hours of incubation, 20. mu.L of MTT solution was added and incubated for 4 hours. Finally, 150. mu.L of DMSO was used instead of the original mixed solution, and the absorbance at 490 nm was read by a microplate reader.

3. Detection of alkaline phosphatase (ALP) activity of hMSCs:

the hMSCs are inoculated into a 6-well culture plate, and cultured for 7 days by using a culture medium added with the GQDs/hydrogel composite nanoparticle leachate. After 7 days, the cells were lysed with a cell lysis buffer, and the lysed sample was centrifuged at 10,000 rpm for 5 min, and the supernatant was collected. The total amount of intracellular protein content, i.e., the absorbance of the reaction solution at 562 nm, was then determined using the BCA protein assay kit. Finally, ALP activity was performed by alkaline phosphatase assay kit.

4. Alizarin red staining and quantitative analysis:

GQDs/hydrogel composite nanoparticles were fixed in a 6-well plate and sterilized with an ultraviolet lamp for 30 minutes. 40 million cells were added to each well and cultured continuously for 14 days with medium changed every three days. To evaluate osteogenic differentiation effect, hMSCs were washed three times with PBS, fixed with 4% paraformaldehyde at room temperature for 30 min, washed three times with PBS, and washed twice with deionized water to remove excess salts. Add 1 mL alizarin red to each well, incubate for 5-10 min at room temperature, and wash three times with PBS. The staining results were photographed with an optical microscope. To quantify the alizarin red staining, 10% cetylpyridinium chloride was added to the cells, and after incubation for 10 min, the absorbance at 570 nm was measured.

5. Constructing a mouse skull defect model:

a5-week mouse was selected as a model, and the mouse was first anesthetized with sodium pentobarbital (40 mg/kg), the head was wiped with physiological salt and the top hair was removed. Then, the head was wiped with a medical alcohol cotton ball, the head was cut along the cross-suture with a scalpel, a defect of about 2 mm in diameter was drilled out on the mouse skull with a bone drill, and the skull debris was removed with a cotton swab and bleeding was stopped with gauze in time during the drilling. The GQDs/hydrogel-loaded composite was injected into the defect site and irradiated with uv light for 10 minutes to form a hydrogel (care was taken to cover the eyes of the mice during irradiation). The osteogenic efficiency of the different hydrogels was assessed using Micro-CT imaging on day 2 and day 30, respectively.

According to a further aspect of the present invention there is provided the use of a hydrogel composite as defined in any one of the above in a scaffold material for bone defect repair.

The invention can produce the beneficial effects that:

1) according to the graphene quantum dot/hydrogel composite material provided by the invention, GQDs with osteogenic differentiation inducing potential are loaded on hydrogel to form the composite material, the defect that the hydrogel can not effectively promote osteogenic differentiation is overcome, the defect that the GQDs can not be condensed and formed is overcome, and the composite material is independently applied to the body as a bone defect repair support, so that a new possible support material is provided for promoting the healing of a bone defect part in the body as soon as possible.

2) The composite bone defect repairing scaffold material prepared by the invention has good osteogenic differentiation promoting effect, has good biocompatibility, is non-toxic, high in safety, simple in preparation process and low in cost, and provides a possible scaffold material for repairing bone defects clinically.

3) The preparation method of the hydrogel composite material provided by the invention is simple and convenient to operate, stable and controllable in reaction and suitable for industrial production.

Drawings

FIG. 1 is a pictorial view of a GQDs/hydrogel composite material.

FIG. 2 shows the cytotoxicity of GQDs/hydrogel composites on hMSCs.

FIG. 3 shows ALP activity after induction of hMSCs by GQDs/hydrogel composites.

FIG. 4 is a graph of alizarin red staining effect and a quantitative graph of mineralized nodule percentage after GQDs/hydrogel composite material treatment for 14 days.

FIG. 5 is a mouse skull Micro-CT image of a mouse skull after a GQDs/hydrogel composite material is implanted into a mouse skull defect model for one month. (the left figure is the hydrogel group without GQDs, the right figure is the hydrogel group with GQDs with different surface charges, and the surface charges of the GQDs from left to right are positive charges, neutral charges and negative charges in sequence).

Detailed Description

The present application will be described in detail with reference to examples, but the present application is not limited to these examples.

Unless otherwise specified, the raw materials and catalysts in the examples of the present application were purchased commercially, in which graphene quantum dots (abbreviated as GQDs) were prepared by an existing microwave method.

Example 1

1. Preparing GQDs/hydrogel composite material:

a) preheating 1 g of gelatin and 10mL of PBS at 50 ℃ for 20 minutes, and then adding 0.8 mL of methacrylic acid and stirring vigorously; preheating PBS for 10 minutes at 40 ℃, and adding 40 mL of PBS into the mixed solution to terminate the reaction;

b) dialyzing the solution at 40-50 deg.C for 3 days;

c) lyophilizing the solution of (c) with a lyophilizer;

Example 2

Culture and Induction of hMSCs

And (3) placing the hMSCs in a basal culture medium for culture, and changing the culture solution once every three days. Until the cell fusion degree reaches 80% -90%, the ratio of the total weight of the cells is 1: 3, and amplifying. After 24 h of incubation, the basal medium is replaced by the bone induction medium, the bone induction medium is used as a blank control, the leachate of the hydrogel without GQDs is used as a control, and the leachate of the hydrogel compound loaded with the GQDs is used as an experimental group.

2. Alizarin red staining and quantitative analysis

For alizarin red s (ars) staining, hMSCs were washed twice with PBS 14 days after induction with hydrogels loaded with GQDs, and then fixed with 4% paraformaldehyde for 30 min at room temperature. Thereafter, the cells were incubated with 0.1% alizarin red solution at room temperature for 10 min, washed twice with PBS and observed using an optical microscope. The samples were air dried and stained with ARS eluting with 10% cetylpyridinium chloride. The solution from each well was then transferred to a 96-well plate and the absorbance was read with a spectrophotometer at an absorbance wavelength of 570 nm. The experiments were repeated three times for each treatment group and averaged to quantitatively measure their level of promotion of stem cell mineralization.

Example 3

1. Constructing a mouse skull defect model:

a5-week mouse was selected as a model, and the mouse was first anesthetized with sodium pentobarbital (40 mg/kg), the head was wiped with physiological salt and the top hair was removed. Then, the head was wiped with a medical alcohol cotton ball, the head was cut along the cross-suture with a scalpel, a defect of about 2 mm in diameter was drilled out on the mouse skull with a bone drill, and the skull debris was removed with a cotton swab and bleeding was stopped with gauze in time during the drilling.

2. Skull defect repair surgery:

GQDs/hydrogel composite nanoparticles were injected into the defect site and irradiated with UV light for 10 minutes to form hydrogel (care was taken to cover the eyes of the mice during irradiation). The osteogenic efficiency of the different hydrogels was evaluated on day 2 and day 30 using Micro-CT.

The composite material prepared in the embodiment is tested by an instrument and is subjected to related experiments such as MTT method determination of cytotoxicity, in-vitro induced stem cell differentiation, in-vivo osteogenic differentiation and the like, and the results are as follows:

1. as can be seen from fig. 2, after the hMSCs are treated with different leachates for 48 h, the cell viability measured by the MTT method shows that there is no significant difference in cell viability among groups regardless of the presence or absence of the loaded quantum dots, and the cell viability of the hMSCs still exceeds 80% after the samples of each group are treated, which indicates that the blank hydrogel and the hydrogel composite material loaded with GQDs have no biotoxicity and good biocompatibility.

2. As can be seen from FIG. 3, the ALP activity of hMSCs treated with different leachates for 7 days is significantly increased compared with that of the control group, which indicates that the hydrogel loaded with GQDs has the ability of significantly promoting the enhancement of the ALP activity.

3. As can be seen from fig. 4, after alizarin red staining, the mineralization levels of hMSCs after 14 days of stimulation with hydrogels containing GQDs were significantly increased compared to the control group, showing a highly dense network of mineralized nodules, which is consistent with the trend of the ALP activity. The mineralization quantification result is shown in fig. 4, and the absorbance value of the hydrogel group containing GQDs at 570 nm is the largest, which indicates that the mineral substance density is the highest and the differentiation effect is the best.

4. As can be seen from FIG. 5, the simple hydrogel hardly contributes to the healing of the skull defect, but rather has a poor osteogenic repair function, resulting in a deformation of the defect. After the hydrogel loaded with GQDs is implanted into a mouse body, obvious bone healing tendency is found, new bones are formed more densely and tend to be formed at the central part of bone defect, and the bone density of some parts is almost the same as that of normal bone tissues. The result shows that the hydrogel provides a good biological scaffold for GQDs, and the hydrogel complements each other to jointly complete the bone tissue repair.

Although the present application has been described with reference to a few embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the application as defined by the appended claims.

Claims

1. The graphene quantum dot/hydrogel composite material is characterized by comprising a cross-linked network and graphene quantum dots adsorbed on the cross-linked network;

2. The graphene quantum dot/hydrogel composite material of claim 1,

the biocompatible macromolecule is selected from at least one of gelatin, chitosan, hyaluronic acid and collagen;

the crosslinking agent is selected from at least one of methacrylic acid, acrylic acid, crotonic acid and xx.

3. The graphene quantum dot/hydrogel composite material of claim 1, wherein the crosslinked network and the graphene quantum dot are adsorbed by hydrogen bonding or electrostatic interaction.

4. The graphene quantum dot/hydrogel composite material according to claim 1, wherein the graphene quantum dot/hydrogel composite material comprises the following components in parts by mass:

biocompatible macromolecules: crosslinking agent = (0.5-1): (0.5-0.8);

cross-linking the network: graphene quantum dots = (300-: (0.1-0.3).

5. The graphene quantum dot/hydrogel composite material of claim 1, wherein the graphene quantum dot/hydrogel composite material is a porous structure.

6. The method for preparing the graphene quantum dot/hydrogel composite material as claimed in any one of claims 1 to 5, comprising the steps of:

b) dialyzing the first solution to obtain a second solution;

c) lyophilizing the second solution to obtain a hydrogel composite precursor;

7. The method for preparing the graphene quantum dot/hydrogel composite material according to claim 6, wherein in step a),

the reaction temperature is 30-50 ℃; the reaction time was 120-180 minutes.

8. The preparation method of the graphene quantum dot/hydrogel composite material according to claim 6, wherein in the step b), the dialysis temperature is 40-50 ℃; the dialysis time is 3-5 days.

9. The method for preparing the graphene quantum dot/hydrogel composite material according to claim 6, wherein in step c),

the mass ratio of the hydrogel composite precursor to the initiator is (1-3): (0.005-0.015);

preferably, the initiator is a photoinitiator;

10. Use of the graphene quantum dot/hydrogel composite material of any one of claims 1 to 5 in a bone defect repair scaffold material.