CN115160444A - Method for preparing hydrogel scaffolds and use of scaffolds obtained thereby - Google Patents

Abstract

The invention relates to a method for preparing a hydrogel scaffold by utilizing self-assembled polypeptide and application of the scaffold obtained by the method, wherein the preparation method comprises the following steps: 1) Bonding the self-assembly peptide sequence with the nerve growth factor mimic peptide through a covalent bond to obtain a bonding functional polypeptide; 2) Separating and culturing macrophages, inducing the macrophages to be 'substitution activated' anti-inflammatory M2 macrophages, obtaining culture supernatant and filtering to obtain filtered cell culture supernatant; 3) And mixing the filtered cell culture supernatant with the bonded functional polypeptide to obtain a mixed solution, adjusting the concentration of the mixed solution, and performing self-assembly on the bonded functional polypeptide to form the hydrogel support. The invention adopts M2 macrophage condition culture supernatant to construct regeneration microenvironment, and combines with the polypeptide functional hydrogel bracket to realize better integral interaction, so as to prepare the hydrogel bracket capable of supplementing and regulating nerve regeneration, and the bracket can promote nerve cell regeneration behavior and provide a new choice for tissue engineering biomaterials.

Description

Method for preparing hydrogel scaffolds and uses of scaffolds obtained therefrom

This application is a divisional application of the patent application with the application number of 202110849059.8 with the application date of July 27, 2021 and the title of "Method for preparing a hydrogel scaffold and use of the scaffold obtained therefrom".

technical field

The present invention relates to the technical field of tissue engineering, and more particularly, to a method for preparing a hydrogel scaffold by using self-assembled polypeptides and the use of the scaffold obtained thereby.

Background technique

The main causes of peripheral nerve injury include traffic accidents, mechanical trauma, natural disasters and surgical accidents. There are about 1 million new cases of peripheral nerve injury in my country every year, and the resulting disability that is difficult to recover seriously affects the quality of life and mental health of patients, and brings a heavy economic burden to the family and society. At the same time, it is known in the art that the repair effect of artificial nerve implants is still far from that of autologous nerves. Improving the function of implants through local regeneration microenvironment regulation is a key issue for clinical application.

The emergence of tissue engineering technology provides a technical way for the repair of damaged nerves. Tissue engineering consists of three elements: scaffolds, seed cells and signaling factors. Among them, scaffolds not only play an important role in physically connecting and supporting the regenerating tissue, but also in regulating the cell regeneration environment. The three-dimensional network structure of hydrogel has the characteristics of high water content and suitable for cell growth, and the cell growth environment is further improved by cross-linking growth factor mimetic peptides. Therefore, how to successfully construct a scaffold material that can well simulate the in vivo microenvironment is very important for It is important for the regeneration of damaged nerves.

Macrophages are widely distributed in the body, play a huge role in regulating the inflammatory process, are highly plastic, and play an important role in the development of the body and the balance of the internal environment. After the macrophages are polarized to the M2 phenotype, the polarized macrophage subtypes can meticulously regulate and respond to various stimuli, secrete signaling factors, resist chronic inflammation, promote tissue repair and regeneration, and have a positive effect on diseased tissues and organs. The degree of repair plays a key role. The research on the construction of hydrogel scaffolds from M2 macrophage-derived extracellular matrix has not been reported yet, which has important research value.

SUMMARY OF THE INVENTION

One aspect of the present invention is to provide a method for preparing a self-assembling polypeptide hydrogel scaffold, wherein the method includes:

(1) The self-assembled peptide sequence is covalently bonded to the nerve growth factor mimetic peptide to obtain a bonded functional polypeptide;

(2) Isolating and culturing macrophages and inducing them to be "alternatively activated" anti-inflammatory M2 macrophages, obtaining cell culture supernatants and filtering them to obtain filtered cell culture supernatants;

(3) mixing the filtered cell culture supernatant with the binding functional polypeptide to obtain a mixed solution and adjusting the concentration of the mixed solution, and the binding functional polypeptide self-assembles to form a hydrogel scaffold.

Preferably, the nerve growth factor mimetic peptide has the function of promoting the regeneration of neurovascular cells.

Preferably, the self-assembling peptide sequence is covalently linked to the nerve growth factor mimetic peptide via an amide bond or a disulfide bond.

Preferably, the molar ratio of the self-assembling peptide sequence to the nerve growth factor mimetic peptide is (1~3):1.

Preferably, the macrophages are derived from one of rat/mouse peritoneal cavity or bone marrow, or human peripheral blood mononuclear lymphocytes.

Preferably, the culturing in step (2) is carried out under the conditions of 30°C to 40°C and 3% to 10% CO ₂ ; further preferably, the culturing process includes: culturing the macrophages in a medium After growing to 60%~80% confluence, discard the culture medium and wash with PBS buffer, then add fresh medium to the washed culture and continue to cultivate for more than 36h, such as 36~72h, preferably 40~ 50h;

Preferably, the cell culture supernatant is filtered using a filter material with a pore size of 0.2-0.45 μm.

Preferably, the method for preparing M2 macrophage culture supernatant is characterized in that the cell culture inducer adopts 10ng/ml IL-4 to conduct the induction in step (2).

Preferably, the polypeptide hydrogel scaffold has a nanofibrous structure.

Preferably, the polypeptide hydrogel scaffold has the form of a hydrogel, the pH of the adjusted gel is 6.9-7.2, and the concentration is 0.5wt%-4wt%.

Another aspect of the present invention is to provide the use of the prepared hydrogel scaffold for culturing neural Schwann cells in vitro, wherein the scaffold promotes the growth and activity of neural Schwann cells.

The use of the multi-hydrogel scaffold prepared by the method of any one of the present invention in tissue engineering regeneration is characterized in that the scaffold promotes regeneration of injured nerves.

The beneficial effects of the present invention are:

1. By chemical modification and self-assembly methods, polypeptide hydrogel scaffolds with clearer components, stronger functions, higher safety and efficiency are prepared. The nanostructures are more suitable for the repair of damaged nerves and enhance the regeneration of specific nerve cells. speed and improve clinical outcomes.

2. Synthesize sequences containing different nerve growth factor mimetic peptides, prepare hydrogels by self-assembly technology, and further synthesize hydrogel scaffolds containing M2 macrophage conditioned culture conditions to promote the development of synergistic therapy of regenerative cells.

3. Different polypeptide carriers and different nerve growth factor mimetic peptides can also be selected to design and prepare a polypeptide hydrogel scaffold for different nerve cells.

4. A suitable regenerative immune environment The bionic scaffold has good nerve cell adhesion and growth performance, suitable histocompatibility, nanofibers are close to the structure of extracellular matrix, and the scaffold prepared by the method of the present invention has a better regenerative microenvironment , laying the foundation for product development.

Description of drawings

FIG. 1 is a schematic diagram showing the structure of a self-assembling peptide sequence bound to a nerve growth factor mimetic peptide by covalent bonds.

Figure 2 is a diagram showing the morphology of induced M2 macrophages under a microscope.

FIG. 3 is a flow cytometric graph showing the maturity of M2 macrophages. Among them, the percent expression of CD206 detected by flow cytometry is shown.

Figure 4 is a transmission electron microscope image showing a polypeptide hydrogel scaffold. Therein, the structure of the nanofibers is shown.

FIG. 5 is a diagram showing a tilt experiment of the polypeptide hydrogel scaffold.

Figure 6 is a morphological diagram of primary cultured neural Schwann cells under microscope.

FIG. 7 is a graph showing the growth rates of neural Schwann cells cultured in a control group and neural Schwann cells cultured on a polypeptide hydrogel scaffold. Wherein, * represents P<0.05.

Detailed ways

Unless otherwise defined, 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. See, eg, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989).

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. In fact, the present invention is not limited to the methods and materials described herein, but various conventional adjustments or modifications can be made based on the spirit of the present invention, and the adjusted or modified solutions still fall within the protection scope of the present invention. Inside.

As used herein, the terms "a" and "an" and the like encompass plural referents unless the context clearly dictates otherwise.

As used herein, what is modified by the term "about" encompasses approximations within error due to measurement error and the like.

In this paper, unless otherwise defined, the term "tumor microenvironment" is used to refer to the internal environment in which tumor cells develop and live, characterized by low oxygen, low pH, and high pressure, which make the tumor microenvironment contain a large number of The immune inflammatory response produced by growth factors, cell chemokines and various proteolytic enzymes is beneficial to tumor proliferation, invasion, adhesion, angiogenesis, and anti-radiotherapy chemotherapy, and promotes the occurrence of malignant tumors.

In one embodiment, the present invention relates to a method for preparing a self-assembling polypeptide hydrogel scaffold, wherein the method comprises:

(1) The self-assembled peptide sequence is covalently bonded to the nerve growth factor mimetic peptide to obtain a bonded functional polypeptide;

(2) Isolating and culturing macrophages and inducing them to be "alternatively activated" anti-inflammatory M2 macrophages, obtaining cell culture supernatants and filtering them to obtain filtered cell culture supernatants;

(3) mixing the filtered cell culture supernatant with the binding functional polypeptide to obtain a mixed solution and adjusting the concentration of the mixed solution, and the binding functional polypeptide self-assembles to form a nanohydrogel scaffold.

In this paper, the self-assembling peptide sequence is self-assembled from glutamic acid, lysine, alanine, tryptophan, arginine, etc. in an aqueous solution to form a small molecule polypeptide with a length of 10-20 amino acids. As a preferred example, the small molecule polypeptide hydrogels include, but are not limited to, RADARADARADARADA, KWKAKAKAKWK, EWEAEAEAE, FEFEFKFKK, and QQKFQFQFEQQ. The above amino acids were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

In a preferred embodiment, the nerve growth factor mimetic peptide can be any one known in the art, for example, from one or more selected from the group consisting of nerve growth factor (NGF), brain-derived nerve trophic factor (BDNF), vascular endothelial growth factor (VEGF), neurotrophic factor-3 (NT-3) and neurotrophic factor-4 (NT-4).

In a preferred embodiment, the self-assembling peptide sequence and the nerve growth factor mimetic peptide are covalently linked by chemical bonds, and the molar ratio is (1-3): 1, for example, from one selected from the group consisting of amide bonds or disulfide bonds. If the amide bond is selected by solid phase synthesis; if the disulfide bond or the polypeptide carrier is selected by epitope peptide modification of cysteine, solid phase synthesis method, then the two peptides are docked.

The macrophages mentioned herein can be one of rat/mouse peritoneal cavity or bone marrow, or human peripheral blood mononuclear lymphocytes, or can be macrophages isolated by conventional means known in the art .

In a preferred embodiment of the present invention, any suitable medium known in the art can be used for culturing the cells (for example, see the following description: http://www.cellbank.org.cn /peiyang.asp; https://www.atcc.org/), such as but not limited to RPMI-1640 medium containing fetal bovine serum, DMEM medium containing fetal bovine serum, F-12 medium containing fetal bovine serum Medium, DMEM/F-12 medium containing fetal bovine serum. In a further preferred embodiment, the culture is carried out under the conditions of 30°C to 40°C and 3% to 10% CO ₂ . In a further preferred embodiment, the culturing process includes: after growing the tumor cells in the culture medium to a confluence of 60%-80%, discarding the culture medium and washing with PBS buffer , then add fresh medium to the washed culture and continue to culture for more than 36h, for example, 36~72h, preferably 40~50h.

In a preferred embodiment, the cell culture supernatant is filtered using a filter material with a pore size of 0.2-0.45 μm. The cell culture supernatant obtained by filtration mainly contains: proteins secreted by cells (including various cytokines that are the main substances affecting the function of immune cells); non-coding RNAs (including small RNAs and long-chain RNAs, etc.); DNA, etc.

In a preferred embodiment, the method for preparing the M2 macrophage culture supernatant is characterized in that the cell culture inducer is selected from IL-4, preferably, the cell culture stimulus is 0.1- 100 ng/mL, eg 10 ng/mL of IL-4. Add medium for culture, any suitable medium known in the art (for example, see the description in the following: http://www.cellbank.org.cn/peiyang.asp; https://www.atcc .org/), such as but not limited to RPMI-1640 medium containing fetal bovine serum, DMEM medium containing fetal bovine serum, F-12 medium containing fetal bovine serum, DMEM/F-12 medium with fetal bovine serum. In a further preferred embodiment, the culture is carried out under the conditions of 30°C to 40°C and 3% to 10% CO ₂ . In a further preferred embodiment, the culturing is performed for more than 36 hours, for example, 36 to 72 hours, preferably 40 to 50 hours.

In a preferred embodiment, the polypeptide hydrogel scaffold is characterized in that the scaffold has a nanofiber structure.

In a preferred embodiment, the polypeptide hydrogel scaffold is characterized in that the scaffold has the form of a hydrogel, and the pH of the gel after adjustment is 6.9-7.2, and the concentration is 0.5 wt %-4 wt %. For example 1wt%.

In a preferred embodiment, each mL of the hydrogel-containing cell suspension contains 1×10 ⁴ to 1×10 ⁷ cells, preferably 5×10 ⁴ to 1×10 ⁶ cells, such as 1×10 cells. ⁵ of the neural Schwann cells.

In a preferred embodiment, the predetermined shape of the support can be a three-dimensional cell culture scaffold, cell culture dish, cell culture flask, cell culture microplate, double-layered cell culture plate or any known in the art suitable for Three-dimensional cell culture system, such as 6-well cell culture microplate, 12-well cell culture microplate, 24-well cell culture microplate, 96-well cell culture microplate, 24-well double-layer cell culture plate, or the like Any cell culture microplate is commercially available, such as the commercial three-dimensional cell culture systems provided by Thermofisher, FlexCell, and the like.

In a preferred embodiment, the 3D cell culture is carried out at 30°C to 40°C and 3% to 10% CO ₂ for 2-72h, such as 3-72h, 6-72h.

In one embodiment, the present invention relates to the use of the above-mentioned polypeptide hydrogel scaffold in the development of damaged nerve repair. For example, the damaged nerve regeneration can be promoted by injecting or coating the polypeptide hydrogel scaffold treated by conventional physical, chemical or biological methods into the nerve repair catheter.

The solution of the present invention will be further illustrated by the following examples, but the protection scope of the present invention is not limited to this.

Example

The following examples are for illustrative purposes only, and are not intended to limit the protection scope of the present application. Unless otherwise stated, all reagents, materials and equipment used in the following examples are commercially available, or can be formulated or obtained according to prior art well known in the art. Unless otherwise specified, the specific experimental methods involved in the following examples are all the existing technologies in the field (for example, "Molecular Cloning Experiment Guide" (4th Edition), edited by J. Sambrook et al., translated by He Fuchu , Science Press, 2017; conventional means recorded in "Medical Immunology" (7th edition), edited by Cao Xuetao, People's Medical Publishing House, 2018).

Example 1 Preparation of self-assembling peptide sequence-bonded nerve growth factor mimetic peptide

The self-assembled peptide sequence and the nerve growth factor mimetic peptide were covalently linked by chemical bonds, the molar ratio was 1:1, and the covalent linkage was selected from amide bonds. As shown in Figure 1, the two peptides were docked by solid-phase synthesis (self- The assembled peptide sequence RADARADARADARADA and the brain-derived neurotrophic factor mimetic peptide GGGIDKRHWNS).

Example 2 Rat peritoneal macrophage culture

Extraction and isolation of rat peritoneal giant thiamine cells: SD rats aged 2 to 3 months (body weight about 300 g) were killed by de-neck method, soaked in 75 vol% ethanol for 10 min, and then lifted upside down, with no Inject 10 mL of DMEM high-glucose basal medium into the abdominal cavity with a syringe of bacteria. After massaging the abdomen of the rat with fingers for 2 min, the rat was placed in a supine position for 7 min, then the rat abdominal cavity was opened aseptically, about 8 mL of abdominal fluid was drawn out with another sterile syringe, centrifuged at 400 g for 10 min, and the supernatant was discarded. The cells were resuspended in 6 mL high-glucose DMEM complete medium and inoculated into T25 culture flasks, incubated at 37°C in a 5% CO ₂ cell incubator, and the medium was changed after 4 h. The adherent cells are the extracted rat peritoneal macrophages.

Example 3 Preparation of M2 macrophage culture supernatant

The obtained macrophages were isolated and cultured in high glucose DMEM complete medium for 3 days at 37°C, 5% CO ₂ , the original medium was discarded and replaced with 10ng/ml IL-4, 10% FBS, 1% double antibody The RMPI 1640 medium was cultured for 24 h to obtain M2 macrophages. During the culture, the cells were observed to grow well. As shown in Figure 2, after taking pictures by confocal microscope, it was observed that the pseudopodia of the cells were obvious and the shape was normal. The cultured M2 macrophages were collected and labeled with CD206 antibody, and the purity of the cultured M2 macrophages was detected by flow cytometry. As shown in Figure 3, after stimulation with high glucose DMEM complete medium containing 10 ng/mL of IL-4 for 3 days, the double-positive expression rate of CD206 antibody can reach more than 70%, and the purity of M2 macrophages is ideal. The M2 macrophage medium was replaced with serum-free high-glucose medium for cultivation, and the supernatant was collected after 24 h, and the cells and debris were filtered off with a 0.22 μm filter for use.

Example 4 Preparation and structural characterization of self-assembled hydrogel scaffolds

The polypeptide sequence obtained in Example 1 was dissolved in the M2-type macrophage supernatant solution obtained in Example 3, and the concentration was adjusted to form a hydrogel through spontaneous self-assembly of the polypeptide. When the mass concentration of the assembly was 10 mg/mL and above, as shown in Figure 4-5, it appears as a hydrogel with a cross-linked three-dimensional network structure. Through transmission electron microscopy, the results show that the length and composition of polypeptide segments have a great influence on the morphology and solution-gel transition temperature of polymer nano-assembly.

Example 5 Rat primary Schwann cell isolation and culture method and 3D cell culture

Take the red-skinned mouse on the ultra-clean bench, spray it with alcohol, wipe the whole body of the mouse tail, and cut off the head. Adjust the hindlimb of the mouse with the right hand, with the center of the paw facing upward, and press the hindlimb and tail of the mouse with the thumb and index finger of the left hand, respectively. The skin on the hind limbs and the upper tail of the mouse was clipped. Use curved tweezers to open a hole in the depression of the hindlimb, cut twice along the spine, separate the meat from the spine, remove the upper meat, and expose the sciatic nerve and other tissues to be taken. The sciatic nerve with SD erythrocytes for 1 day was placed in a petri dish with high glucose DMEM complete medium. Gently aspirate the supernatant and remove the sciatic nerve into an EP tube (5 mL). Add 1 mL of collagenase (20 pieces) to digest for 30 minutes, quickly cut the tissue with ophthalmic scissors, gently pipette and mix well and put it in an incubator (37°C, 5% CO ₂ ). After 30 minutes, add an equal amount of 1 mL of 0.25% trypsin (Try), gently pipetting and mixing, and put it in the incubator for 5 minutes. After 5 min, add at least three times high glucose DMEM complete medium to stop digestion (3-4 times), blow evenly, centrifuge at 1200 rpm for 5 min, and discard the supernatant. Add fresh high-glucose DMEM complete medium, blow well, filter, and seed cells into petri dishes. On the second day, the medium was changed 16 hours after the inoculation, and the cells were cultured with high glucose DMEM complete medium containing cytarabine (1:1000). The medium was changed every four days and cultured with high DMEM complete medium until confluent. The collected Schwann cells were mixed with the above-mentioned hydrogel scaffold according to the volume ratio of 1:3 respectively, and the cell concentration was adjusted to 1×10 ⁵ cells/mL, and incubated at 37°C and 5% CO ₂ for 30 minutes. , to obtain each cell suspension containing the gel.

Embodiment 6 Activity detection of Schwann cell fraction

After CFSE labeling, Schwann cells were cultured in high glucose DMEM complete medium for 3 days at 37°C and 5% CO ₂ , and the proliferation of Schwann cells was detected by flow cytometry. The results are shown in FIG. 6 . Observe that the cells are spherical, and more cells show protrusions, which can better simulate the regenerative microenvironment. Compared with the control group, the growth rate of Schwann cells in the hydrogel scaffold group was significantly increased by flow cytometry.

Claims (7)

1. A bonding functional polypeptide comprising a self-assembling peptide sequence and a nerve growth factor mimetic peptide bonded by a covalent bond, wherein the self-assembling peptide sequence is RADARADARADARADA, KWKAKAKAKWK, EWEAEAEAE, FEFEFKFKK or QQKFQFQFEQQ, and the The nerve growth factor mimetic peptide is GGGIDKRHWNS. 2 . The functional bonding polypeptide of claim 1 , wherein the self-assembling peptide sequence and the nerve growth factor mimetic peptide are covalently linked through an amide bond or a disulfide bond. 3 . The bonding function polypeptide according to claim 1 or 2, wherein the molar ratio of the self-assembling peptide sequence to the nerve growth factor mimetic peptide is (1~3):1. 4. The use of the functionally bonded polypeptide according to any one of claims 1-3 in the preparation of a hydrogel scaffold, wherein the functionally bonded polypeptide is in the cell culture supernatant of anti-inflammatory M2 macrophages The hydrogel scaffold is formed by self-assembly in liquid. 5. The use of claim 4, wherein "alternatively activated" anti-inflammatory M2 macrophages are induced by isolating and culturing macrophages from: rat/mouse peritoneal cavity or bone marrow , or one of human peripheral blood mononuclear lymphocytes. 6. The use of claim 5, wherein the induction is carried out with 10 ng/ml of IL-4. 7. The use of any one of claims 4-6, wherein the scaffold has a nanofibrous structure.

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