Piezoelectric electrospinning rod compounded collagen-based hydrogel as well as preparation method and application thereof

Abstract

The invention discloses piezoelectric electrospinning rod compounded collagen-based hydrogel as well as a preparation method and application thereof, and belongs to the field of piezoelectric biological materials. Carrying out electrospinning on polyhydroxybutyrate through an electrospinning technology to prepare piezoelectric oriented fibers loaded with piezoelectric ceramics; preparing the piezoelectric oriented fiber into a piezoelectric electrospinning rod by using a freezing slicing technology; collagen and chitosan are cross-linked by using a biological cross-linking agent to form collagen-based hydrogel; and compounding the piezoelectric electrospinning rod in the collagen-based hydrogel to prepare the collagen-based hydrogel compounded by the piezoelectric electrospinning rod. The hydrogel prepared by the invention has good piezoelectric property, better mechanical property and excellent biocompatibility, and can well meet the requirement of an endogenous electric field at a cartilage defect.

Description

Technical Field

[0001] The present invention belongs to the field of piezoelectric biomaterials, specifically a piezoelectric electrospinning rod composite collagen-based hydrogel and its preparation method and application. It has good piezoelectric properties, mechanical properties and biocompatibility and can restore the endogenous electric field to promote the repair of cartilage defects. Background Art

[0002] Cartilage tissue is nerve-free and blood-free. After injury, it cannot effectively deliver nutrients and related growth factors for self-repair. In severe cases, it can develop into osteoarthritis, causing great pain, mobility problems, and even disability to patients, seriously affecting their quality of life. Therefore, external means must be used to promote cartilage repair. Endogenous electric fields are widely distributed in the human body, not only in nerve tissue, but also in skin tissue, bone and cartilage tissue, etc. They can regulate the cell cycle and promote cell proliferation, migration, differentiation and tissue regeneration. Therefore, correcting the endogenous electric field disorder caused by tissue injury through electroactive biomaterials is a new treatment strategy. As an emerging smart material, piezoelectric materials will spontaneously generate electric fields when subjected to external mechanical stimulation, effectively avoiding the drawbacks of traditional electrical stimulation therapy, and are favored by more and more researchers in the field of tissue repair. Summary of the Invention

[0003] The present invention aims to prepare a piezoelectric electrospinning rod-composite collagen-based hydrogel with good piezoelectric properties, mechanical properties and biocompatibility and the ability to restore the endogenous electric field and promote the repair of cartilage defects.

[0004] The present invention first discloses the following technical solutions, including:

[0005] (1) Preparation of piezoelectric oriented fibers: piezoelectric oriented fibers loaded with piezoelectric ceramics were prepared by electrospinning polyhydroxybutyrate;

[0006] (2) Preparation of piezoelectric electrospinning rods: piezoelectric oriented fibers were prepared into piezoelectric electrospinning rods using cryosectioning technology;

[0007] (3) Preparation of collagen-based hydrogel: using a biological cross-linking agent to cross-link collagen and chitosan to form a collagen-based hydrogel;

[0008] (4) Loading piezoelectric electrospinning rods: piezoelectric electrospinning rods are composited into collagen-based hydrogels to prepare piezoelectric electrospinning rod-composite collagen-based hydrogels.

[0009] Furthermore, the piezoelectric oriented fiber in step (1) is prepared by the following method:

[0010] First, 0.4-0.8 parts by mass of polyhydroxybutyrate was dissolved in hexafluoroisopropanol and stirred for 4-8 hours to obtain a blank electrospinning solution with a mass fraction of 4-8%. Then, 40-80% of piezoelectric ceramic particles were added and vigorously stirred at 1000-1500 rpm for 8-12 hours to obtain an electrospinning solution loaded with piezoelectric ceramics. The electrospinning parameters were then adjusted to produce oriented fibers loaded with piezoelectric ceramics. The electrospinning process parameters included a voltage of 12-18 kV, a receiving distance of 12-18 cm, and a spinning solution feed rate of 0.5-2 mL / h. The spinning environment was maintained at a temperature of 25±6°C and a humidity of 30±10%.

[0011] Furthermore, the piezoelectric ceramics described in step (1) include one or more combinations of zinc oxide, barium titanate, strontium titanate, strontium carbonate, and potassium sodium niobate.

[0012] Furthermore, the piezoelectric electrospinning rod in step (2) is prepared by the following method:

[0013] First, the oriented electrospun membrane loaded with piezoelectric ceramics was folded perpendicular to the direction of the electrospun fibers to a length of 1-2 cm and a width of 0.6-1.2 cm. The membrane was then placed in an embedding cassette and embedded in 50%-100% OCT embedding medium. After the membrane was fully infiltrated, it was frozen in liquid nitrogen for 5-10 minutes and then cut using a cryostat set to 20-40 μm. The electrospun rod was then collected and repeatedly rinsed with deionized water until no foam was visible, and then freeze-dried to obtain the piezoelectric electrospun rod.

[0014] Furthermore, the collagen-based hydrogel in step (3) is prepared by the following method:

[0015] 1 mass fraction of chitosan was dissolved in 2% acetic acid solution to obtain a 1% chitosan solution, followed by adding a 1% collagen solution and stirring for 8 to 12 hours under ice bath conditions. The pH was then adjusted to 4 to 7 with 2M sodium hydroxide, a biocrosslinker was added, and crosslinking was performed at room temperature for 12 to 24 hours to obtain the target collagen-based hydrogel.

[0016] Furthermore, the biocrosslinking agent in step (3) includes one or more combinations of glutaraldehyde, genipin, and tannic acid.

[0017] Furthermore, the mass fraction of the biocrosslinking agent in step (3) is 0.2% to 0.6%.

[0018] Furthermore, in step (3), the mass ratio of chitosan to collagen is 1:1-4.

[0019] Furthermore, the mass fraction of the piezoelectric electrospinning rods in the collagen-based hydrogel described in step (4) is 0.2% to 0.6%.

[0020] The present invention also discloses the use of a piezoelectric electrospinning rod-compounded collagen-based hydrogel prepared by any of the above preparation methods in the preparation of cartilage repair materials.

[0021] Compared with the prior art, the advantages of the present invention are:

[0022] (1) The present invention uses electrospinning technology to load nano-zinc oxide onto polyhydroxybutyrate fibers, effectively enhancing the hydrogel's ability to locally generate an electric field and its mechanical properties;

[0023] (2) The present invention uses cryosectioning technology to prepare electrospun fibers into electrospun rods, effectively solving the problem that electrospun membranes cannot be evenly dispersed in the hydrogel system;

[0024] (3) The present invention compensates for the poor mechanical properties of collagen-based hydrogels by utilizing the rigidity of chitosan, and the two form a more stable polyelectrolyte composite hydrogel network with better mechanical properties through electrostatic interaction, which can slow down the enzymatic hydrolysis rate of collagen and make the degradation rate of the hydrogel more in line with the needs of tissue regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a SEM photograph of the unloaded piezoelectric ceramic particle aligned fibers of the embodiment;

[0026] Figure 2 This is a SEM photograph of the piezoelectric ceramic oriented fibers with a relative mass fraction of 60% in the embodiment;

[0027] Figure 3 This is the SEM of piezoelectric ceramic oriented fibers with a relative mass fraction of 80%;

[0028] Figure 4 This is a SEM photo of the electrospinning rod of the embodiment;

[0029] Figure 5 This is one of the SEM photos of the piezoelectric electrospinning rod composite hydrogel in the embodiment;

[0030] Figure 6 This is the second SEM photo of the piezoelectric electrospinning rod composite hydrogel of the embodiment;

[0031] Figure 7 Graph showing the piezoelectric performance of the piezoelectric electrospinning rod composite hydrogel of the embodiment;

[0032] Figure 8 Graph showing the mechanical properties of the piezoelectric composite hydrogel of the embodiment;

[0033] Figure 9 This is a graph showing the degradation of the piezoelectric composite hydrogel of the embodiment within 21 days;

[0034] Figure 10 This is a graph showing the blood compatibility of the piezoelectric composite hydrogel according to the embodiment. DETAILED DESCRIPTION

[0035] The specific technical solutions of the present invention are described with reference to the embodiments.

[0036] Example 1:

[0037] (1) Preparation of piezoelectric oriented fibers: First, 0.6 mass fractions of polyhydroxybutyrate were dissolved in hexafluoroisopropanol and stirred for 8 hours to obtain a 6 mass fraction blank electrospinning solution. Then, 40%-80% of piezoelectric ceramic particles were added and vigorously stirred at 1500 rpm for 12 hours to obtain an electrospinning solution loaded with piezoelectric ceramics. Then, by adjusting the electrospinning parameters, oriented fibers loaded with piezoelectric ceramics were prepared, wherein the electrospinning process parameters were: voltage 16 kV, receiving distance 18 cm, spinning solution feed rate 1 mL / h; spinning environment conditions were temperature 25°C, humidity 35%. The piezoelectric ceramic was zinc oxide, and the mass fraction of the piezoelectric ceramic relative to polyhydroxybutyrate was 60%.

[0038] (2) Preparation of piezoelectric electrospinning rods: First, the oriented electrospun membrane loaded with piezoelectric ceramics was folded perpendicular to the electrospun fiber direction to a length of 1.5 cm and a width of 1 cm. The membrane was then placed in an embedding box and embedded with 50% OCT embedding medium. After the membrane was fully infiltrated, it was frozen with liquid nitrogen for 5 minutes and then cut using a cryostat with the parameters set to 25 μm. The electrospun rods were then collected and repeatedly washed with deionized water until no obvious foam was observed. The piezoelectric electrospinning rods were then freeze-dried to obtain the obtained rods.

[0039] (3) Preparation of piezoelectric collagen-based composite hydrogel: 1 mass fraction of chitosan was dissolved in 2% acetic acid solution to obtain a 1% chitosan solution, which was then added to a piezoelectric electrospinning rod and stirred overnight to mix. The mixture was then stirred with a 1% collagen solution in an ice bath for 12 h. The pH was then adjusted to 4.5 with 2M sodium hydroxide, and a biocrosslinker was added. The target hydrogel was crosslinked at room temperature for 16 h. The biocrosslinker was genipin with a mass fraction of 0.5%; the mass ratio of chitosan to collagen was 1:2; and the mass fraction of the piezoelectric electrospinning rod was 0.4%.

[0040] Example 2:

[0041] (1) Preparation of piezoelectric oriented fibers: First, 0.6 mass fractions of polyhydroxybutyrate were dissolved in hexafluoroisopropanol and stirred for 6 hours to obtain a blank electrospinning solution with a mass fraction of 6%. Then, piezoelectric ceramic particles with a relative mass fraction of 40%-80% were added and vigorously stirred at 1000 rpm for 8 hours to obtain an electrospinning solution loaded with piezoelectric ceramics, wherein the mass fraction of the piezoelectric ceramics relative to polyhydroxybutyrate was 60%. Then, by adjusting the electrospinning parameters, oriented fibers loaded with piezoelectric ceramics were prepared, wherein the electrospinning process parameters were: voltage 14 kV, receiving distance 15 cm, spinning solution feed rate 0.8 mL / h; spinning environment conditions were temperature 27°C and humidity 40%. The piezoelectric ceramic was barium titanate, and the mass fraction of the piezoelectric ceramic relative to polyhydroxybutyrate was 80%.

[0042] (2) Preparation of piezoelectric electrospinning rods: First, the oriented electrospun membrane loaded with piezoelectric ceramics was folded perpendicular to the electrospun fiber direction to a length of 2 cm and a width of 1.5 cm. The membrane was then placed in an embedding box and embedded with an 80% OCT embedding medium. After the membrane was fully infiltrated, it was frozen with liquid nitrogen for 10 minutes and then cut using a cryostat with the parameters set to 20 μm. The electrospun rods were then collected and repeatedly washed with deionized water until no obvious foam was observed. The target electrospun rods were then freeze-dried to obtain the desired electrospun rods.

[0043] (3) Preparation of piezoelectric collagen-based composite hydrogel: 1 mass fraction of chitosan was dissolved in 2% acetic acid solution to obtain a 1% chitosan solution, which was then added to a piezoelectric electrospinning rod and stirred overnight to mix. The mixture was then stirred with a 1% collagen solution in an ice bath for 8 h. The pH was then adjusted to 5 with 2M sodium hydroxide, and a biocrosslinker was added. The target hydrogel was crosslinked at room temperature for 12 h. The biocrosslinker was glutaraldehyde with a mass fraction of 0.2%; the mass ratio of chitosan to collagen was 1:4; and the mass fraction of the piezoelectric electrospinning rod was 0.2%.

[0044] The present invention provides the following experimental data, which are all experimental results obtained based on Example 1:

[0045] Figures 1 to 4 The following are SEM photos of electrospun oriented fibers and electrospinning rods. Figure 1 is unloaded piezoelectric ceramic particles, Figure 2 For piezoelectric ceramics with a relative mass fraction of 60%, Figure 3 For piezoelectric ceramics with a relative mass fraction of 80%, Figures 1 to 3 It can be seen that by adjusting the electrospinning parameters, neatly arranged oriented fibers were successfully prepared, with the diameter concentrated around 1 μm, and with the decrease of the piezoelectric ceramic content, the orientation of the electrospun fibers tended to be consistent; Figure 2It can be seen that ideal electrospinning rods were successfully prepared using the cryosectioning technique, and their lengths were all around 25 μm, which was consistent with the set parameters.

[0046] Figure 5 and Figure 6 This is a SEM image of the piezoelectric composite hydrogel. It shows a three-dimensional network structure with a loose, porous structure that facilitates the exchange of nutrients and metabolic waste. A partial magnification shows the successful loading of the previously prepared electrospinning rod, providing the conditions for generating an output voltage.

[0047] Figure 7 This is the piezoelectric performance diagram of piezoelectric hydrogel.

[0048] Col-CS@0ER, Col-CS@2ER, Col-CS@4ER, and Col-CS@6ER represent electrospinning rod loadings in the hydrogel at 0 mg / ml, 2 mg / ml, 4 mg / ml, and 6 mg / ml, respectively. This performance was measured using an exciter coupled with a multimeter. The output voltage was recorded at a 10 Hz exciter frequency. The output voltage graph shows that the output voltage increases with increasing electrospinning rod loading, demonstrating that the resulting electric field meets the requirements for cartilage repair.

[0049] Figure 8 is the mechanical properties diagram of piezoelectric composite hydrogel; Figure 9 The degradation diagram of piezoelectric composite hydrogel within 21 days. Figure 8 It can be seen that as the content of electrospinning rods increases, the mechanical strength of the piezoelectric composite hydrogel increases, but when the electrospinning rod content reaches 6 mg / ml, it shows a weakening trend. It is speculated that the reason may be that the excessive content of electrospinning rods destroys the three-dimensional network structure of the hydrogel, resulting in a certain degree of cracking. Figure 9 It can be seen that the piezoelectric composite hydrogel still retained 20% of its mass after 21 days of degradation, and the hydrogel as a whole remained intact. It can be seen that the introduction of chitosan enhanced the cross-linking network of the hydrogel, making the degradation rate of the collagen-based hydrogel more in line with the needs of tissue regeneration.

[0050] Figure 10 The hemocompatibility graph of the piezoelectric composite hydrogel shows that the hemolysis rate of the prepared piezoelectric hydrogel is less than 5%, meeting the international biomaterial hemocompatibility standards and demonstrating good biosafety.

[0051] Those skilled in the art will appreciate that the embodiments described herein are intended to help readers understand the implementation methods of the present invention, and it should be understood that the scope of protection of the present invention is not limited to such specific descriptions and embodiments. Those skilled in the art can make various other specific variations and combinations based on the technical teachings disclosed in the present invention without departing from the essence of the present invention, and such variations and combinations are still within the scope of protection of the present invention.

Claims

1. A method for preparing a collagen-based hydrogel composited with a piezoelectric electrospinning rod, characterized in that: The following steps are involved: (1) Preparation of piezoelectric oriented fibers: piezoelectric oriented fibers loaded with piezoelectric ceramics were prepared by electrospinning polyhydroxybutyrate; (2) Preparation of piezoelectric electrospinning rods: piezoelectric oriented fibers were prepared into piezoelectric electrospinning rods using cryosectioning technology; (3) Preparation of collagen-based hydrogel: using a biological cross-linking agent to cross-link collagen and chitosan to form a collagen-based hydrogel; (4) Loading piezoelectric electrospinning rods: piezoelectric electrospinning rods are composited into collagen-based hydrogels to prepare piezoelectric electrospinning rod-composite collagen-based hydrogels.

2. The method for preparing a piezoelectric electrospinning rod composite collagen-based hydrogel according to claim 1, characterized in that: The specific method of step (1) is: First, polyhydroxybutyrate was dissolved in hexafluoroisopropanol and stirred to obtain a blank electrospinning solution. Then, piezoelectric ceramic particles were added and stirred vigorously to obtain an electrospinning solution loaded with piezoelectric ceramics. Then, by adjusting the electrospinning parameters, piezoelectric oriented fibers loaded with piezoelectric ceramics were prepared.

3. The method for preparing a piezoelectric electrospinning rod composite collagen-based hydrogel according to claim 2, characterized in that: The method of step (1) is based on the following parameters: The materials are in the following proportions: 0.4-0.8 parts by weight of polyhydroxybutyrate is dissolved in hexafluoroisopropanol to obtain a blank electrospinning solution with a mass fraction of 4-8%; piezoelectric ceramic particles with a relative mass fraction of 40%-80% are added; The electrospinning process parameters are: The voltage is 12-18 kV, the receiving distance is 12-18 cm, and the spinning solution feeding rate is 0.5-2 mL / h. The spinning environment conditions are a temperature of 25±6° C. and a humidity of 30±10%.

4. The method for preparing a piezoelectric electrospinning rod composite collagen-based hydrogel according to claim 2, characterized in that: The piezoelectric ceramic particles described in step (1) are one or more combinations of zinc oxide, barium titanate, strontium titanate, strontium carbonate, and potassium sodium niobate.

5. The method for preparing a piezoelectric electrospinning rod composite collagen-based hydrogel according to claim 1, characterized in that: The specific method of step (2) is: First, the piezoelectric oriented fiber membrane was folded along the direction perpendicular to the electrospun fiber with a length of 1-2 cm and a width of 0.6-1.2 cm, placed in an embedding box, and embedded with an OCT embedding agent with a concentration of 50%-100%. After the OCT embedding agent fully infiltrates the electrospun membrane, it was frozen with liquid nitrogen for 5-10 minutes. Then, a cryo-microtome was used to cut the piezoelectric electrospinning rods at a parameter of 20 to 40 μm; The piezoelectric electrospinning rods were collected and repeatedly washed with deionized water until no obvious foam was found, and then freeze-dried.

6. The method for preparing a piezoelectric electrospinning rod composite collagen-based hydrogel according to claim 1, characterized in that: The specific method of step (3) is: 1 mass fraction of chitosan was dissolved in 2% acetic acid solution to obtain a 1% chitosan solution, followed by adding a 1% collagen solution and stirring for 8 to 12 hours under ice bath conditions. The pH was then adjusted to 4 to 7 with 2M sodium hydroxide, and a biocrosslinking agent was added. The mixture was cross-linked at room temperature for 12 to 24 hours to obtain a collagen-based hydrogel.

7. The method for preparing a piezoelectric electrospinning rod composite collagen-based hydrogel according to claim 6, characterized in that: The biological cross-linking agent includes: one or more of glutaraldehyde, genipin, and tannic acid; The mass fraction of the biological cross-linking agent is 0.2% to 0.6%; the mass ratio of chitosan to collagen is 1:1 to 4.

8. The method for preparing a piezoelectric electrospinning rod composite collagen-based hydrogel according to claim 6, characterized in that: The mass fraction of the piezoelectric electrospinning rod in the collagen-based hydrogel in step (4) is 0.2% to 0.6%.

9. A piezoelectric electrospinning rod-composite collagen-based hydrogel obtained according to the preparation method according to any one of claims 1 to 9.

10. The piezoelectric electrospinning rod-compounded collagen-based hydrogel according to claim 9 is used as a raw material for preparing cartilage repair materials.

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