CN112972767B

CN112972767B - Magnetic response tissue engineering material with osteogenesis promoting effect and preparation method and application thereof

Info

Publication number: CN112972767B
Application number: CN202110162250.5A
Authority: CN
Inventors: 叶招明; 黄东骅; 徐恺成; 黄鑫; 林秾; 俞小华
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2021-02-05
Filing date: 2021-02-05
Publication date: 2022-03-11
Anticipated expiration: 2041-02-05
Also published as: CN112972767A

Abstract

The invention discloses a magnetic response tissue engineering material with an effect of promoting osteogenesis, a preparation method and application thereof, wherein the preparation method of the material comprises the following steps: firstly, adjusting the pH value and osmotic pressure of a collagen I solution, then sequentially adding an aminated ferroferric oxide magnetic particle solution and a crosslinking agent genipin solution according to a certain proportion, uniformly mixing by shaking after adding the solution every time, and finally incubating for at least 3 hours in a constant-temperature incubator at 37 ℃ to obtain the genipin crosslinked ferroferric oxide magnetic particle collagen I hydrogel. The material can change the appearance of the internal fiber structure of the material in a time sequence under the action of an external magnetic field, thereby having the capability of directionally inducing macrophage to polarize to M2 and promoting the osteogenesis of a bone defect part, and being used for researching the influence of a tissue engineering material on an osteogenesis immune microenvironment and the mechanism thereof.

Description

Magnetic response tissue engineering material with osteogenesis promoting effect and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicine materials. More particularly, relates to a magnetic response tissue engineering material with an effect of promoting osteogenesis, and a preparation method and application thereof.

Background

The large-section bone defect is usually caused by trauma, bone tumor excision and serious infection, has long treatment period, high difficulty, more complications and high disability rate, and is a great technical difficulty to be urgently solved in clinics of orthopedics department. Currently, common treatment methods include autologous bone grafting, allogeneic bone grafting and bone tissue engineering. The autologous bone has limited material availability, cannot meet the requirement of bone transplantation with large defect amount, and can cause new trauma to the bone supplying area. The allograft bone transplantation has the problems of transplant rejection, easily-transmitted diseases, high bone non-healing rate and the like. Therefore, the treatment of large bone defects by bone tissue engineering means is a future trend and is a hot spot and a difficult point of current clinical research, and a tissue engineering material with high-efficiency bone defect repairing effect is eagerly designed.

The core of bone tissue engineering is mainly focused on seed cells, growth factors and scaffold materials. Of particular interest are scaffold materials that provide temporary physical support to cells and tissues while providing a microenvironment conducive to cell growth and differentiation. Currently, most materials are dedicated to induce differentiation of stem cells into osteogenic direction, so as to repair bone defects, and good osteogenic effect is observed in vitro experiments. However, recent studies have shown that the immune system plays a key regulatory role in the osteogenesis process. The local proinflammatory microenvironment at the fracture site greatly hinders the osteogenic effect of stem cells. Therefore, the conclusions of in vitro experiments did not take into account in vivo immune microenvironment factors. How to design a material can regulate and control the development of a local immune microenvironment to the direction beneficial to bone formation, or can greatly promote the in-vivo bone formation effect of the material.

Macrophages, a major effector cell in the immune system, are highly localized around bone tissue and widely distributed. Studies have shown that macrophages play a key role in the formation of the immune microenvironment surrounding bone tissue: macrophages can be polarized into different functional phenotypes under different environmental stimuli, mainly comprising M1 type and M2 type, wherein M1 type can aggravate inflammation and remove fragments at fracture broken ends, and M2 type macrophagy polarization is proved to promote osteogenic differentiation of stem cells and further promote bone repair. Furthermore, there is a literature report that the duration of M1-mediated inflammation has a major effect on bone formation, that transient acute inflammation is beneficial, that transient inflammatory phase followed by a regenerative phase dominated by M2 may have optimal bone formation, and that premature suppression of immune responses (e.g. non-steroidal anti-inflammatory drugs or glucocorticoid drugs) may lead to suboptimal bone regeneration. Thus, modulating macrophage polarization in a timely manner may be a strategic means of promoting bone healing.

Disclosure of Invention

In order to overcome the defects that the fiber structure arrangement of the existing tissue engineering material (such as hydrogel and the like) is disordered, the polarization ability of macrophages to M2 cannot be regulated and controlled in real time according to experimental requirements and the like, the invention provides a novel genipin crosslinked ferroferric oxide magnetic particle collagen I hydrogel magnetic response tissue engineering material.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a preparation method of a magnetic response tissue engineering material with an effect of promoting osteogenesis comprises the following steps:

firstly, adjusting the pH value and osmotic pressure of the collagen I hydrogel, then sequentially adding an aminated ferroferric oxide magnetic particle solution and a crosslinking agent genipin solution according to a certain proportion, uniformly mixing by shaking after adding the solution every time, and finally incubating for at least 3-6 hours in a constant-temperature incubator at 37 ℃ to obtain the genipin crosslinked ferroferric oxide magnetic particle collagen I hydrogel.

In the above technical scheme, further, the pH value of the collagen I solution is adjusted by adopting a sodium hydroxide solution and a hydrochloric acid solution, and the osmotic pressure of the collagen I solution is adjusted by adopting a phosphate buffer solution.

Further, the mass ratio of the aminated ferroferric oxide magnetic particles to the collagen I is 1: 160-400.

Further, the mass ratio of the dosage of the crosslinking agent genipin to the collagen I is 1: 16-40.

The invention also provides a magnetic response tissue engineering material with the effect of promoting osteogenesis, which is prepared by adopting the method.

In the preparation process, the magnetic particles and the hydrogel fibers are stably crosslinked through genipin, so that the problem that the magnetic particles on the material fall off is not easy to occur in the subsequent use process; meanwhile, according to experimental requirements, magnetic fields are applied at different stages in a time sequence manner, so that the appearance and tension of hydrogel fibers are changed, and the polarization of macrophages to M2 type is favorably and directionally induced in a regeneration period, and the local osteogenesis effect is promoted.

The material prepared by the method can change the appearance of the internal fibrous structure under the action of an external magnetic field in time sequence according to experimental needs, so that the material has the capability of directionally inducing macrophage to polarize to M2, promotes the osteogenesis of a bone defect part, and provides an effective and feasible technology for researching the influence of tissue engineering materials on an osteogenesis immune microenvironment and mechanism exploration thereof.

The invention principle of the invention is as follows:

if a magnetic response tissue engineering material can be designed, the giant phagocytic M2 polarization can be directionally induced in a time sequence, and a local immune microenvironment is regulated and controlled to be beneficial to stem cell osteogenesis, so that the magnetic response tissue engineering material is likely to have a good bone tissue repair effect. It has been shown that the more ordered the fibers in the material is to favor polarization to M2, and that the cross-linking agent genipin has been shown in vivo experiments to favor polarization to M2. Therefore, the genipin cross-linked ferroferric oxide magnetic particle collagen I hydrogel is formed by cross-linking aminated ferroferric oxide magnetic particles onto collagen I fibers by using a cross-linking agent genipin on the basis of the most common collagen I in bone tissues with good biocompatibility and in a physiological state.

The invention has the beneficial effects that:

the genipin cross-linked ferroferric oxide magnetic particle collagen I hydrogel prepared by the invention can improve a local immune microenvironment (directionally induces macrophage M2 polarization) under the action of a magnetic field, thereby providing a favorable environment for host stem cells to exert the osteogenesis effect, and the hydrogel can be applied to in vivo bone defect repair.

According to the invention, by applying a magnetic field to the tissue engineering material in a time sequence manner, the appearance and the tension of a fiber structure in the material are changed, so that the local immune microenvironment of bone defect is regulated, macrophage M2 polarization is induced in an oriented manner, and the osteogenesis effect is increased.

Drawings

FIG. 1: a flow chart for preparing tissue engineering materials;

FIG. 2: characterization statistical graphs of iron contents of the dry sample and the wet sample of the material before and after the magnetic field is applied;

FIG. 3: magnetization curves of materials at different magnetic particle densities;

FIG. 4: schematic diagram of magnet mold for in vitro culture; wherein 4(a) is a schematic diagram of a magnetic field and a matched mould thereof, and is composed of two magnets (the magnetic field intensity is 0.28-0.32 Tesla) and an adaptive ya-Ke transparent frame positioned between the two magnets; FIG. 4(b) is a customized magnet mold-adapted specialized cell culture plate with a diameter per well equivalent to the diameter of a conventional 24-well plate; FIG. 4(c) is a schematic view of the in vitro magnetic field mold after the special cell culture plate is placed;

FIG. 5: immunofluorescence confirms that the material can induce macrophage M2 polarization;

FIG. 6: ELISA confirms that the material can induce macrophage M2 polarization;

FIG. 7: the macrophage conditioned medium induced by the material can promote the osteogenesis of stem cells;

FIG. 8: statistical results of the osteogenesis of stem cells promoted by material-induced macrophage conditioned medium.

Detailed Description

The instrument comprises the following steps: an aseptic operating platform, a constant-temperature incubator at 37 ℃ and a vortex mixer.

The main reagents are as follows: a collagen I solution (corning, rat tail collagen I, high concentration (8-11mg/mL in 0.02N (gram equivalent/liter) acetic acid), cat # 354249); secondly, an aminated ferroferric oxide magnetic particle solution (Donna biology, PEG ferroferric oxide magnetic nanoparticles (amino terminal), with the cargo number of Mag3300, 10mL, and the Fe content of 10 mg); thirdly, genipin powder (aladdin of alatin, original manufacturer goods number G101204, 25mg, 98%); tetra, 10x phosphate buffered saline (10 x PBS); fifthly, 1mol/L sodium hydroxide solution, 0.1mol/L sodium hydroxide solution and 0.1mol/L hydrochloric acid solution; sixthly, C57BL/6 mouse primary macrophages; seventhly, primary culture medium of macrophage; eighthly, cell climbing (phi 14 mm); ninth, magnets and matching moulds (note: magnets are arranged on both sides of the acrylic transparent frame matched with the magnets, the size of the magnetic field between the moulds is 0.28-0.32 Tesla), and special cell culture plates matched with the moulds (customized, the bottom area of each hole is equal to the area of a single hole of a 24-hole plate); tenthly, a bacteria filter; eleven, EP tube (2 ml); twelfth, tweezers and tissue scissors; thirteen, precision pH paper (6.0-8.0).

Fig. 1 is a schematic flow chart of a preparation method of the magnetic response tissue engineering material with osteogenesis promoting effect, and the specific operation steps are as follows:

step 1 preparation of reagents: adding 1ml of 1 Xphosphate buffer saline (PBS) into 0.01g of genipin powder to prepare 1% genipin solution, placing in a constant-temperature incubator at 37 ℃ to be dissolved in the dark for 3 days, filtering the bacteria by a bacteria filter after complete dissolution, and storing in a refrigerator at 4 ℃ in the dark; filtering 10xPBS buffer solution, 1mol/L sodium hydroxide (NaOH) solution, 0.1mol/L NaOH solution and 0.1mol/L hydrochloric acid (HCl) solution by using a bacteria filter, and then placing the bacteria filter in a refrigerator at 4 ℃ for later use; the collagen I solution is directly placed in a refrigerator at 4 ℃ for standby. The special cell culture plate, the EP tube, the tweezers and the tissue scissors are placed on a sterile operation table for standby after high-pressure steam sterilization, and the precise PH test paper is placed on a super clean bench for ultraviolet irradiation for 30 minutes for sterilization.

Step 2, pH value adjustment: clamping an EP tube by using tweezers, taking 800 mu L of 200-mul collagen I solution by using a liquid transfer gun according to experimental requirements, placing the solution into the EP tube, adding 7.7 mu L of 1mol/L NaOH solution into 300 mu L of the collagen I solution according to empirical values, shaking and uniformly mixing the solution by using a vortex mixer until the collagen I solution is changed into milky turbid from clear and transparent, taking 2 mu L of the solution to be dropped on pH test paper, observing the color change of the reaction of the test paper within 0.5 second, wherein the target pH value is 7.3-7.5, and the reaction of the test paper is green. If the test paper has a bluish reaction color, namely the pH value is higher, adding 2-8 mu L0.1mol/L HCl according to specific conditions; if the reaction color of the test paper is yellow, namely the pH value is low, adding 2-8 mu L0.1mol/L NaOH solution according to specific conditions; and then shaking and mixing uniformly by using a vortex mixer, and repeating the step of measuring the pH until the target pH value range (6.5-7.8) is reached, wherein the collagen I solution is changed into milky collagen I solution from original clear and transparent. All the above operations are performed in a sterile operating station.

Step 3, adjusting electrolyte balance: adding the collagen I pre-glue solution after PH adjustment in the step 2 into the collagen I pre-glue solution according to the ratio of 10: adding 10XPBS buffer solution in a volume ratio of 1 to adjust the electrolyte balance in the hydrogel, so that the osmotic pressure of the hydrogel is similar to that of normal extracellular matrix (approximately equal to that of the 1 XPBS buffer solution), and facilitating the subsequent cell growth; shaking and mixing by using a vortex mixer. All the above operations are performed in a sterile operating station.

Step 4, adding magnetic particles: and (3) adding 3-5% volume ratio of aminated ferroferric oxide magnetic particle solution into the collagen I solution after the electrolyte balance is adjusted in the step (3) according to experimental needs, and shaking and uniformly mixing the solution by using a vortex mixer, wherein the collagen I solution is changed from the original milky white color to a light brown turbid shape, so that the collagen I solution containing aminated ferroferric oxide magnetic particles can be obtained. All the above operations are performed in a sterile operating station.

Step 5, adding a cross-linking agent: adding the amino ferroferric oxide magnetic particles into the collagen I solution containing the amino ferroferric oxide magnetic particles obtained in the step 4 according to the ratio of 100:3 to 100: adding a prepared 1% genipin solution in a volume ratio of 5, and uniformly mixing by shaking with a vortex mixer to obtain a genipin crosslinked ferroferric oxide magnetic particle collagen I pre-prepared solution. This step is carried out under protection from light. All the above operations are performed in a sterile operating station.

And 6, incubation: and (3) placing the genipin crosslinked ferroferric oxide magnetic particle collagen I pre-prepared glue solution obtained in the step (5) into a constant-temperature incubator at 37 ℃ for incubation for 3 hours, wherein the color of the hydrogel is changed from original light brown to dark blue gel, so that genipin crosslinked ferroferric oxide magnetic particle collagen I hydrogel is formed, and the genipin crosslinked ferroferric oxide magnetic particle collagen I hydrogel is the tissue engineering material.

FIG. 2 is a statistical chart of the iron content characterization of the dry and wet samples before and after the magnetic field is applied, which shows that the iron content of the material before and after the magnetic field is applied has no significant change.

Fig. 3 is a graph of the magnetization of materials at different magnetic particle densities, showing that as the magnetic particle density increases, the magnetization capacity increases.

FIG. 4 is a schematic diagram of the magnetic response tissue engineering material used in vitro cell experiments, which can be used for exploring the regulation and control effect of genipin cross-linked ferroferric oxide magnetic particle collagen I hydrogel on local immune microenvironment of bone defect and osteogenesis effect thereof under the action of a time-sequence externally applied magnetic field.

The magnetic response tissue engineering material prepared by the invention has the capability of directionally inducing macrophage to polarize to M2 under the action of an external magnetic field, and the inventor guesses that the capability is probably related to the change of collagen fibers from the original relatively disordered state to the relatively ordered state. Further research shows that M2 macrophages generated by the hydrogel under the action of a magnetic field can efficiently promote osteogenic differentiation of in vitro stem cells.

Example 1

2D culture of macrophages: soaking the cell slide (phi 14mm) in 95% ethanol solution for 30 min for sterilization, clamping with forceps, air drying in a clean bench, and placing into special cell culture plate; shearing a small section of the tip of a pipette tip with the specification of 1ml or 200 mu (determined according to the amount of the actually added gel), taking 300 mu L of the genipin crosslinked ferroferric oxide magnetic particle collagen I hydrogel obtained in the step 6 from the sheared pipette tip, placing the hydrogel on a cell climbing sheet in a special cell culture plate, and incubating the hydrogel in a cell culture box at 37 ℃ for 3 hours until the hydrogel forms gel on the surface of the cell climbing sheet; and then adding 10-100 ten thousand primary macrophages to the surface of the material of each hole according to the actual experiment requirement, adding a proper amount of primary macrophage culture medium, placing the mixture in an in-vitro culture magnetic field mold shown in figure 4, and placing the mixture in a constant-temperature incubator at 37 ℃ for culture, so as to provide subsequent experimental operations of macrophage polarization, immunofluorescence staining and the like. All the above operations are performed in a sterile operating station.

Example 2

3D culture of macrophages: the cell slide (phi 14mm) is soaked in 95% ethanol solution, clamped by tweezers, and naturally dried in a super clean bench, and then placed in a special cell culture plate for later use. And (3) putting the genipin crosslinked ferroferric oxide magnetic particle collagen I pre-colloid solution which is not incubated in the step (5) into a cell culture box at 37 ℃ for incubation for 30 minutes, wherein the material is slightly light blue gel-like (is preliminarily gelatinized and is not shaped). Adding primary macrophage suspension into an EP tube filled with the material according to the volume ratio of 1:1 according to the proportion that 10-100 ten thousand primary macrophages are added into every 100-300 mu L of the material; shearing a small section of the tip of the pipette tip by using tissue scissors, stretching the sheared pipette tip into an EP tube, gently blowing and beating for a plurality of times to fully and uniformly mix the cell suspension and the pre-preg solution, sucking 300 mu L of materials with the same treatment by using a clean pipette tip to a cell climbing sheet in a special cell culture plate, and incubating for 30 minutes in a constant-temperature incubator at 37 ℃ to ensure that the cells grow adherent as much as possible. And then adding a proper amount of macrophage primary culture medium according to actual experiment requirements, placing the macrophage primary culture medium in an in-vitro culture magnetic field mold shown in figure 4, and placing the macrophage primary culture medium in a constant-temperature incubator at 37 ℃ for culture so as to be used for subsequent experiment operations of macrophage polarization, immunofluorescence staining and the like. All the above operations are performed in a sterile operating station.

FIG. 5 shows that immunofluorescence confirms that the material can induce polarization of macrophage M2. And (5) Col I: a collagen I hydrogel; g-Col I: genipin cross-linked collagen I hydrogel; 3%: genipin cross-linked ferroferric oxide magnetic particle collagen I hydrogel with the concentration of 3%; 5%: genipin cross-linked 5% concentration ferroferric oxide magnetic particle collagen I hydrogel; DAPI: cell nucleus staining; CD 206: staining of M2 type macrophages. The most CD206 signal was observed at a magnetic particle concentration of 3%, indicating that genipin cross-linked ferriferrous oxide magnetic particle collagen I hydrogel with a concentration of 3% is beneficial for macrophage polarization to M2.

FIG. 6 shows that ELISA confirms that the material can induce polarization of macrophage M2, and statistics show that when the concentration of ferroferric oxide particles is 3%, the highest concentration of interleukin-10 (IL-10) is measured, and IL-10 is closely related to the polarization of macrophages to M2 type; in addition, after the crosslinking agent genipin is added, the concentration of IL-10 secreted by cells is increased compared with that of a blank control group and collagen I hydrogel without the crosslinking agent; while the secreted tumor necrosis factor alpha (TNF-alpha) between the groups of the material was not significantly different.

FIG. 7 shows that material-induced macrophage conditioned medium promotes stem cell osteogenesis. The figure shows that the osteogenic effect is the best when the concentration of the ferroferric oxide particles is 3%, and the osteogenic effect of genipin cross-linked collagen I hydrogel (g-Col I) and genipin cross-linked ferroferric oxide magnetic particle collagen I hydrogel (5%) with the concentration of 5% is better than that of a Blank control group (Blank) and a pure collagen I hydrogel group (Col I) by virtue of osteogenic staining of stem cells cultured by macrophage conditioned medium.

Fig. 8 is a statistical view of quantitative analysis of the osteogenesis effect of fig. 7, which is shown by measuring the content of calcium (Ca) in each group using a spectrophotometer, and it can be seen that the osteogenesis effect is the best when 3% of the magnetic particles are used.

Claims

1. A preparation method of a magnetic response tissue engineering material with an effect of promoting osteogenesis is characterized by comprising the following steps: firstly, adjusting the pH value and osmotic pressure of a collagen I solution, then adding an aminated ferroferric oxide magnetic particle solution, uniformly vibrating, finally adding a crosslinking agent genipin solution, uniformly vibrating, and incubating in a constant-temperature incubator at 37 ℃ for 3-6 hours to obtain the genipin crosslinked ferroferric oxide magnetic particle collagen I hydrogel.

2. The method for preparing a magnetically responsive tissue engineering material having an osteogenesis promoting effect according to claim 1, wherein the pH of the collagen I solution is adjusted using a sodium hydroxide solution and a hydrochloric acid solution, and the osmotic pressure of the collagen I solution is adjusted using a phosphate buffered saline solution.

3. The method for preparing a magnetic response tissue engineering material with an effect of promoting osteogenesis according to claim 1, wherein the mass ratio of the aminated ferroferric oxide magnetic particles to collagen I is 1: 160-400.

4. The method for preparing a magnetic response tissue engineering material with an effect of promoting osteogenesis according to claim 1, wherein the mass ratio of the dosage of the cross-linking agent genipin to the collagen I is 1: 16-40.

5. A magnetically responsive tissue engineering material having osteogenesis promoting effect, prepared by the method of any one of claims 1 to 4.

6. The use of the magnetically responsive tissue engineering material with osteogenesis promoting effect of claim 5, wherein the morphology of the internal fiber structure is changed in time sequence under the action of the external magnetic field according to experimental needs, so that the magnetically responsive tissue engineering material has the ability of directionally inducing macrophage polarization to M2, promotes osteogenesis of bone defect sites, and can be used for studying the influence of the tissue engineering material on osteogenesis immune microenvironment and mechanism thereof.