CN115160444A

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

Info

Publication number: CN115160444A
Application number: CN202210998757.9A
Authority: CN
Inventors: 杨鹏翔; 杨宇民; 接晶; 张鲁中
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2021-07-27
Filing date: 2021-07-27
Publication date: 2022-10-11
Also published as: CN113713175A; CN113713175B

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 use of scaffolds obtained thereby

The present application is a divisional application of patent application No. 202110849059.8, entitled "method of producing hydrogel scaffold and use of the scaffold obtained thereby", filed on 27/7/2021.

Technical Field

The invention relates to the technical field of tissue engineering, in particular to a method for preparing a hydrogel scaffold by utilizing self-assembled polypeptide and application of the scaffold.

Background

The main causes of peripheral nerve injury include traffic accidents, mechanical trauma, natural disasters, and surgical accidents. About 100 million cases of peripheral nerve injury are newly increased every year in China, the irradiated disability which is difficult to recover seriously affects the life quality and the mental health of patients, and heavy economic burden is brought to families and society. Meanwhile, the repair effect of the artificial nerve implant is known to be different from that of the autonomic nerve in the field, and the key problem of realizing clinical application is that the function of the implant is improved by local regeneration microenvironment regulation.

The emergence of tissue engineering technology provides a technical mode for repairing damaged nerves. Tissue engineering includes three elements: scaffold, seed cells and signaling factors. The scaffold plays an important role in not only physically connecting and supporting regenerated tissues, but also regulating and controlling cell regeneration environment. The three-dimensional network structure of the hydrogel has the characteristics of high water content and suitability for cell growth, and the cell growth environment is further improved by crosslinking the growth factor mimic peptide, so how to successfully construct the scaffold material capable of well simulating the in-vivo microenvironment has important significance for injured nerve regeneration.

Macrophages are extremely widespread in the body, play a great role in regulating inflammation, have high plasticity, and play an important role in the development of the body and the balance of the internal environment. After the macrophages are polarized into M2 phenotype, the polarized macrophage subtype can finely regulate and respond to various different stimuli, secrete signal factors, resist chronic inflammation, promote tissue repair and regeneration and play a key role in the repair degree of diseased tissues and organs. The research on the construction of the hydrogel scaffold by using the extracellular matrix derived from the M2 macrophage is not reported, and the hydrogel scaffold has important research value.

Disclosure of Invention

One aspect of the present invention provides a method for preparing a self-assembled polypeptide hydrogel scaffold, wherein the method comprises:

(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 to obtain cell 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 a hydrogel support.

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

Preferably, the self-assembling peptide sequence is covalently linked to the nerve growth factor mimetic peptide by 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 to 3): 1.

preferably, the macrophage is from one of rat/mouse peritoneal cavity or bone marrow, or human peripheral blood mononuclear lymphocytes.

Preferably, the culture in the step (2) is carried out at 30-40 ℃ and 3-10% CO ₂ Under the conditions of (a); further preferably, the culturing process comprises: growing the macrophages in a culture medium to reach 60-80% confluence, discarding the culture solution, washing the macrophages by using a PBS (phosphate buffer solution) buffer solution, adding a fresh culture medium into the washed culture, and continuously culturing for more than 36 hours, such as 36-72h, preferably 40-50h;

preferably, the cell culture supernatant is filtered by a filter material with the aperture of 0.2 to 0.45 mu m.

Preferably, the method for preparing a culture supernatant of M2 macrophage cell, wherein said cell culture inducer uses IL-4 of 10ng/ml to perform said inducing of step (2).

Preferably, the polypeptide hydrogel scaffold has a nanofibrous structure.

Preferably, the polypeptide hydrogel stent has a hydrogel form, the pH of the gel after adjustment is 6.9 to 7.2, and the concentration is 0.5wt% -4wt%.

Still another aspect of the present invention is to provide a use of the hydrogel scaffold prepared as described above in vitro culture of neuroscheynes cells, wherein the scaffold promotes the growth and activity of the neuroscheynes cells.

Use of a polyhydrogel scaffold prepared by the method of any of the present invention in tissue engineering regeneration, wherein the scaffold promotes regeneration of damaged nerves.

The beneficial effects of the invention are:

1. the polypeptide hydrogel scaffold prepared by the chemical modification and self-assembly method has more definite components, stronger functionality, higher safety and efficiency, and the nano structure is more suitable for repairing damaged nerves, enhancing the regeneration speed of specific nerve cells and improving the clinical treatment effect.

2. Synthesizing sequences containing different nerve growth factor mimic peptides, preparing hydrogel by a self-assembly technology, and further synthesizing a hydrogel scaffold containing an M2 macrophage conditioned culture condition to promote the development of the synergistic treatment direction of regenerative cells.

3. Different polypeptide carriers and different nerve growth factor mimic peptides can be selected, and the polypeptide hydrogel scaffold can be designed and prepared aiming at different nerve cells.

4. The bionic scaffold for regenerating immune environment has good nerve cell adhesion and growth performance and suitable histocompatibility, the nanofiber is close to an extracellular matrix structure, and the scaffold prepared by the method has a better regeneration microenvironment, so that a foundation is laid for developing products.

Drawings

FIG. 1 is a schematic diagram showing a structure in which a self-assembly peptide sequence is covalently bonded to a nerve growth factor mimetic peptide.

Figure 2 is a microscopic morphogram showing induced M2 macrophages.

Figure 3 is a flow cytometric map showing M2 macrophage maturation. Therein, the percentage of expression of CD206 detected by flow cytometry is shown.

Fig. 4 is a transmission electron micrograph showing a polypeptide hydrogel scaffold. Therein, the structure of the nanofibers is shown.

Fig. 5 is a graph showing an experimental tilt of a polypeptide hydrogel scaffold.

FIG. 6 is a morphological diagram of primary cultured neuroschwann cells under a microscope.

Fig. 7 is a graph showing the growth rates of the neuregulin cells cultured by the control group and the neuregulin cells cultured by the polypeptide hydrogel scaffold. Wherein, represents P <0.05.

Detailed Description

Unless defined otherwise, 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, e.g., 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 can 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 modifications and adaptations can be made based on the spirit of the present invention, and the modified or adapted solution still falls within the scope of the present invention.

As used herein, the terms "a" and "an" and the like encompass a plurality of the subject matter unless the context clearly dictates otherwise.

In this document, the object modified by the term "about" covers approximate values within the error range due to measurement errors and the like.

As used herein, unless otherwise defined, the term "tumor microenvironment" refers to the internal environment of the genesis and life of tumor cells, and has the characteristics of hypoxia, low pH and high pressure, which allow the tumor microenvironment to have a large number of immunoinflammatory responses generated by growth factors, cytokines and various proteolytic enzymes, thereby facilitating the proliferation, invasion, adhesion, angiogenesis and anti-radiation chemotherapy of tumors and promoting the generation of malignant tumors.

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

(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 nano-hydrogel support.

Herein, the self-assembly peptide sequence is formed by self-assembly of glutamic acid, lysine, alanine, tryptophan, arginine and the like in an aqueous solution to form a small molecule polypeptide with the length of 10-20 amino acids. As preferred examples, the small molecule polypeptide hydrogels include, for example, but are not limited to, RADARADARADADA, KWKAKAKAKAKWK, EWEAEAE, FEFEFKFKK, and QQKFQQFEQEQQ. The above amino acids were purchased from sigma aldrich trade ltd.

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

The macrophage as referred to herein may be one from rat/mouse peritoneal cavity or bone marrow, or human peripheral blood mononuclear lymphocytes, or may be a macrophage isolated by conventional means known in the art.

In a preferred embodiment of the invention, the culturing of the cells may be performed using any suitable medium known in the art (see, for example, the following description: http:// www.cellbank.org.cn/peeyang.asp; https:// www.atcc.org /), such as, but not limited to, RPMI-1640 medium with fetal bovine serum, DMEM medium with fetal bovine serum, F-12 medium with fetal bovine serum, DMEM/F-12 medium with fetal bovine serum. In a further preferred embodiment, the culture is carried out at 30-40 ℃ and 3-10% CO ₂ Under the conditions of (1). In a further preferred embodiment, the culturing comprises: growing said tumor cells in said mediumAfter reaching 60-80% confluence, the culture solution is discarded and washed by PBS buffer solution, and then fresh culture medium is added into the washed culture and the culture is continuously cultured for more than 36 hours, such as 36-72h, preferably 40-50h.

In a preferred embodiment, the cell culture supernatant is filtered using a filter material having a pore size of 0.2 to 0.45. Mu.m. The cell culture supernatant obtained by filtration mainly contains: proteins secreted by cells (including various cytokines as main substances affecting immune cell functions); non-coding RNA (including small RNA and long-chain RNA); DNA, and the like.

In a preferred embodiment, the method for preparing a culture supernatant of M2 macrophages, wherein the cell culture inducer is selected from IL-4, preferably wherein the cell culture stimulant is IL-4 in an amount of 0.1-100 ng/mL, such as 10 ng/mL. The culture is carried out in any suitable medium known in the art (see, for example, the 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, DMEM/F-12 medium containing fetal bovine serum). In a further preferred embodiment, the culture is carried out at 30-40 ℃ and 3-10% CO ₂ Under the conditions of (1). In a further preferred embodiment, the culture is carried out for 36 hours or more, for example, 36 to 72h, preferably 40 to 50h.

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 stent is characterized in that the stent has a hydrogel form, the pH of the gel after adjustment is 6.9 to 7.2, and the concentration is 0.5wt% to 4wt%, for example 1wt%.

In a preferred embodiment, there is 1X 10 per mL of said hydrogel-containing cell suspension ⁴ ~1×10 ⁷ Preferably 5X 10 ⁴ ~1×10 ⁶ Over, e.g. 110 ⁵ And the schwann cells.

In a preferred embodiment, the support of predetermined shape may be a three-dimensional cell culture scaffold, a cell culture dish, a cell culture flask, a cell culture microplate, a bilayer cell culture plate or any system known in the art suitable for three-dimensional cell culture, such as a 6-well cell culture microplate, a 12-well cell culture microplate, a 24-well cell culture microplate, a 96-well cell culture microplate, a 24-well bilayer cell culture plate or any cell culture microplate commercially available in the art, such as the commercialized three-dimensional cell culture systems provided by Thermofisher, flexCell, etc.

In a preferred embodiment, the 3D cell is cultured at 30-40 ℃ and 3-10% CO ₂ For 2 to 72h, for example for 3 to 72h, for 6 to 72h.

In one embodiment, the present invention relates to the use of the polypeptide hydrogel scaffold described above for the development of repair of damaged nerves. For example, the regeneration of damaged nerves is promoted by injecting, coating, etc. a polypeptide hydrogel scaffold treated by a conventional physical, chemical or biological method into a nerve repair catheter.

The invention is further illustrated by the following examples, without limiting the scope of the invention thereto.

Examples

The following examples are for illustrative purposes only and are not intended to limit the scope of the present application. Unless otherwise indicated, all reagents, materials and equipment used in the examples below were either commercially available or could be formulated or obtained according to prior art techniques well known in the art. Unless otherwise stated, specific experimental means referred to in the following examples are conventional means described in the prior art in the field (for example, molecular cloning guide (4 th edition), edited by j. Sambrook et al, congratulatory translation, scientific press, 2017, medical immunology (7 th edition), edited by caoko tao press, public health press, 2018).

EXAMPLE 1 preparation of self-assembling peptide sequence-bonded nerve growth factor mimetic peptide

Covalently linking the self-assembly peptide sequence with the nerve growth factor mimic peptide through a chemical bond, wherein the molar ratio is 1:1, covalent linkage selected from amide bonds, as shown in FIG. 1, two peptides were docked by solid phase synthesis (self-assembling peptide sequence RADARADARADARADA and brain-derived neurotrophic factor mimetic peptide GGGIDKRWNS).

Example 2 rat peritoneal macrophage culture

Extracting and separating rat celiac giant thia cells: SD rats of 2-3 months of age (about 300g in body weight) are sacrificed by cervical dislocation, soaked in 75vol% ethanol for 10 min, then the rats are inverted and lifted, and 10 mL of DMEM high-sugar base medium is injected into the abdominal cavity of the rats by a sterile syringe. After 2 min of rubbing with fingers, rats were kept in a supine position for 7 min, then the abdominal cavity of rats was aseptically opened, approximately 8 mL of the abdominal cavity liquid was extracted with another sterile syringe, centrifuged at 400 g for 10 min, the supernatant was discarded, cells were resuspended in 6 mL of high-glucose DMEM and inoculated into a T25 flask at 37 ℃ with 5% CO ₂ And (5) incubating in a cell incubator, and changing the solution after 4 hours. The adherent cells are the macrophages of the abdominal cavity of the extracted rat.

EXAMPLE 3 preparation of M2 macrophage culture supernatant

Separating the obtained macrophage and keeping the macrophage at 37 deg.C and 5% CO ₂ Then, after culturing in high-sugar DMEM complete medium for 3 days, the original medium was discarded, and the medium was replaced with 10ng/ml IL-4, 10% FBS, 1% double antibody RMPI 1640 medium, and the culture was continued for 24 hours to obtain M2-type macrophages. The growth state of the cells was observed to be good during the culture. As shown in FIG. 2, the pseudopodia of the cells is obvious and the morphology is normal when the cells are photographed by a confocal microscope. The cultured M2-type macrophages are collected and labeled with CD206 antibody, and the purity of the cultured M2-type macrophages is checked by flow cytometry. As shown in FIG. 3, after the complete culture medium of high-glucose DMEM containing 10ng/mL of IL-4 is adopted for stimulation for 3 days, the double positive expression rate of the CD206 antibody can reach more than 70%, and the purity of M2 type macrophage is ideal. The M2 type macrophage culture medium is replaced by a serum-free high-sugar culture medium for culture, and after 24 hours, the supernatant is collected and filtered by a 0.22 mu M filter to remove cells and fragments for standby.

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, the concentration was adjusted, and a hydrogel was formed by spontaneous self-assembly of the polypeptide, and when the mass concentration of the assembly was 10mg/mL or more, the assembly exhibited a hydrogel and a crosslinked three-dimensional network structure, as shown in FIGS. 4 to 5. The results of a transmission electron microscope show that the length and the components of the polypeptide chain have great influence on the nano-assembly morphology and the solution-gel transition temperature of the polymer.

Example 5 isolation and culture method of rat Primary Schwann cells and 3D cell culture

Taking the red skin mouse, spraying alcohol on the red skin mouse, wiping the whole body of the tail of the mouse, and cutting off the head. The hind limb of the mouse is adjusted by the right hand, the paw heart is upward, and the hind limb and the tail of the mouse are respectively pressed by the thumb and the forefinger of the left hand. The skin of the mouse was cut from the hind limb and above the tail. A bent forceps is used for clamping an opening in the depression area of the hind limb, two times of spine scissors are cut, meat is separated from the spine, the upper meat is removed, and tissues to be taken, such as sciatic nerves and the like, are exposed. Sciatic nerves of SD erythroderma cells were taken for 1 day and placed in a culture dish of high-glucose DMEM complete medium. The supernatant was gently aspirated off, and the sciatic nerve was removed and placed in an EP tube (5 mL). Adding collagenase 1mL (20 pieces), digesting for 30min, rapidly shearing tissue with ophthalmic scissors, gently blowing, mixing, and placing into incubator (37 deg.C, 5% CO) ₂ ). After 30min, the mixture is gently blown and beaten by equal amount of 1mL of 0.25% pancreatin (Try) and is put in an incubator for 5min. After 5min, add at least three times high-glucose DMEM complete medium, stop digestion (3-4 times), blow well, centrifuge at 1200rpm for 5min, discard supernatant. Adding fresh high-sugar DMEM complete culture medium, blowing uniformly, filtering, and inoculating cells into a culture dish. The next day, the culture was changed 16h after inoculation and cultured in high-sugar DMEM complete medium containing cytarabine (1. The medium was changed every four days and cultured in complete high DMEM medium until full. Uniformly mixing the collected Schwann cells and the hydrogel scaffold according to the volume ratio of 1 ⁵ one/mL, 5% CO at 37 ℃ ₂ Incubated for 30 minutes to obtain gel-containing cellsAnd (3) suspending the cells.

Example 6 Activity assay of Schwann cell fraction

After CFSE labeling, schwann cells were incubated at 37 ℃ in 5% CO ₂ Then, after culturing in a high-glucose DMEM complete medium for 3 days, the proliferation of schwann cells was detected by a flow cytometer. The results are shown in fig. 6. Cells are observed to be spherical, more cells show processes, and therefore, the regeneration microenvironment can be better simulated. The growth speed of the Schwann cells of the hydrogel bracket group is obviously improved compared with that of a control group through detection of a flow cytometer.

Claims

1. A linked functional polypeptide comprising a self-assembling peptide sequence and a nerve growth factor mimetic peptide, wherein said self-assembling peptide sequence is RADARADARADARADA, KWKAKAKAKWK, ewaeaeae, fefefefkk, or qqkfqfeqq, and said nerve growth factor mimetic peptide is GGGIDKRHWNS, linked by a covalent bond.

2. The linked functional polypeptide of claim 1, wherein the self-assembling peptide sequence is covalently linked to the nerve growth factor mimetic peptide by an amide bond or a disulfide bond.

3. The linkage functional polypeptide of claim 1 or 2, wherein the molar ratio of the self-assembling peptide sequence to the nerve growth factor mimetic peptide is (1 to 3): 1.

4. use of the bonded functional polypeptide of any of claims 1-3 in the preparation of a hydrogel scaffold, wherein the bonded functional polypeptide self-assembles in the cell culture supernatant of an anti-inflammatory M2 macrophage to form the hydrogel scaffold.

5. The use according to claim 4, wherein the anti-inflammatory M2 macrophage cell is induced to be "surrogate activated" by isolating and culturing macrophages from: rat/mouse peritoneal cavity or bone marrow, or human peripheral blood mononuclear lymphocytes.

6. Use according to claim 5, wherein said induction is carried out with 10ng/ml of IL-4.

7. Use according to any one of claims 4 to 6, wherein the scaffold has a nanofibrous structure.