CN112978741A

CN112978741A - Manganese silicate hollow nanosphere capable of immunoregulation vascularization and preparation method and application thereof

Info

Publication number: CN112978741A
Application number: CN202110141779.9A
Authority: CN
Inventors: 吴成铁; 吴金福
Original assignee: Shanghai Institute of Ceramics of CAS
Current assignee: Shanghai Institute of Ceramics of CAS
Priority date: 2021-02-02
Filing date: 2021-02-02
Publication date: 2021-06-18
Anticipated expiration: 2041-02-02
Also published as: CN112978741B

Abstract

The invention relates to a manganese silicate hollow nanosphere capable of immunoregulation vascularization and a preparation method and application thereof. The manganese silicate hollow nanospheres are brown manganese ore phases, have hollow structures and have a chemical composition of (Mn)₂O₃)₃MnSiO₃(ii) a Preferably, the diameter of the manganese silicate hollow nanosphere is 90-100 nm, and the specific surface area is 630-640 m²/g。

Description

Manganese silicate hollow nanosphere capable of immunoregulation vascularization and preparation method and application thereof

Technical Field

The invention relates to a manganese silicate hollow nanosphere capable of immunoregulation vascularization, a preparation method and application thereof, in particular to a manganese silicate hollow nanosphere capable of immunoregulation vascularization, a preparation method thereof, a modified culture medium containing the manganese silicate hollow nanosphere, and a biological ink containing the manganese silicate hollow nanosphere and capable of being used for biological printing.

Background

Angiogenesis is an indispensable key process in the repair of tissues such as bone defects and skin wounds, and is also a great challenge for clinical medicine. The traditional autograft or allograft treatment scheme has the problems of insufficient donor source, secondary wound generation and the like, and ignores the complex environment of the human body, particularly immune rejection and the effect on vascularization. The biological 3D printing technology has the functions of simultaneously printing cells and materials and accurately controlling cell distribution, and can construct a living cell co-culture support simulating an immune microenvironment in vitro. Therefore, the development of the biological ink for biological 3D printing and with immunoregulation vascularization has great practical significance.

Disclosure of Invention

The invention aims to provide manganese silicate hollow nanospheres capable of immunoregulation vascularization and a preparation method and application thereof, wherein the manganese silicate hollow nanospheres are mainly applied to obtain a biological ink containing the manganese silicate hollow nanospheres capable of immunoregulation vascularization, inorganic components (manganese silicate hollow nanospheres) in the biological ink can promote vascularization through immunoregulation, and the biological ink can construct a living cell co-culture scaffold in vitro through biological 3D printing.

In a first aspect, the present invention provides a manganese silicate hollow nanosphere, which is a brown manganese ore phase having a hollow structure and a chemical composition of (Mn)₂O₃)₃MnSiO₃(ii) a Preferably, the silicic acidThe diameter of the manganese hollow nanosphere is 90-100 nm, and the specific surface area is 630-640 m²/g。

In a second aspect, the present invention provides a method for preparing the manganese silicate hollow nanosphere, comprising: dissolving the silicon dioxide nanospheres in deionized water and CH₃COONa (or Na)₂SO₄) And MnCl₂.4H₂Adding triethanolamine or ethanolamine into the mixed solution of O until the pH value is 7.3-7.6; and then carrying out hydrothermal reaction at 100-120 ℃ for 22-26 hours, and finally calcining at 600-700 ℃ for 5-7 hours to obtain the manganese silicate hollow nanospheres.

Preferably, the particle size of the silicon dioxide nanospheres is 100-120 nm; the silica nanosphere and CH₃The mass ratio of COONa is (1-3): 1; the silicon dioxide nanospheres and MnCl₂.4H₂The mass ratio of O is (0.5-2): 1.

in the disclosure, the precursor mesoporous silica of the hollow manganese silicate nanosphere belongs to a silicon-based biomaterial, has excellent bioactivity, and has very large application potential in tissue engineering such as repair of hard tissues and soft tissues; and manganese is a metal element necessary for maintaining various important physiological functions of the human body. The manganese silicate hollow nanospheres prepared by introducing manganese element into mesoporous silica have uniform size, particle diameter of 90-100 nm and specific surface area of 630-640 m²Has excellent degradation performance, bioactivity and immunoregulation function.

The manganese-doped mesoporous silica promotes biodegradation and decomposition due to the addition of manganese, and further accelerates the fracture of Si-O-Si bonds in the framework. The manganese silicate hollow nanospheres synthesized based on the well-dispersed mesoporous silica can quickly release Mn ions and Si ions into a culture medium.

In a third aspect, the present invention provides a modified medium comprising: basic culture medium, and the manganese silicate hollow nanospheres dispersed in the basic culture medium; the basic culture medium comprises the following components: DMEM high-sugar medium, fetal calf serum, penicillin/streptomycin; the concentration of the manganese silicate hollow nanosphere is less than 100 mug/mL.

The conventional method of inducing blood vessels is to add growth factors, but it is difficult to maintain the activity for a long time and the cost is high. In the method, the inorganic manganese silicate is used, and the biological active ions Mn ions and Si ions released by the inorganic manganese silicate are utilized to replace growth factors, so that the remarkable immunoregulation function and the angiogenesis promoting function of the Mn ions are exerted.

Preferably, the concentration of the hollow manganese silicate nanospheres in the modified culture medium is 25-100 μ g/mL.

In the modified culture medium, when the concentration of the manganese silicate hollow nanosphere is below 100 mu g/mL, the biological compatibility is good. For example, concentrations of 25. mu.g/mL and 100. mu.g/mL both promoted macrophage polarization to exhibit the anti-inflammatory M2 phenotype and increased expression of the angiogenic factor VEGF. And the concentration of the extract is higher than 100 mu g/mL, so that the extract has an inhibition effect on macrophage proliferation by cells.

The culture medium generated by the interaction of the manganese silicate hollow nanospheres and the macrophages is taken as a conditioned medium. The endothelial cells are cultured by using the conditioned medium, and the immune microenvironment formed by the manganese silicate hollow nanospheres with the concentration of 25-100 mug/mL and macrophages is proved to activate a VEGF passage in the endothelial cells, so that angiogenesis is facilitated.

In a fourth aspect, the present invention provides a novel bio-ink comprising a phosphate buffer solution, and a bioactive inorganic component and a biocompatible organic component dispersed in the phosphate buffer solution; the biological active inorganic component is the manganese silicate hollow nanosphere, and the biological compatible organic component is at least two of sodium alginate, hyaluronic acid, collagen, methyl cellulose and carrageenan; the mass ratio of the bioactive inorganic component to the biocompatible organic component is (0-0.2): 13.

in the field, most of traditional biological ink components are organic substances, and only compatibility with cells can be ensured, and the components lack functional effects and have limited clinical effects. Therefore, in the invention, inorganic bioactive ions are additionally introduced into the bio-ink containing the biocompatible organic component, so that the bio-ink is endowed with the function of immunoregulation into blood vessels, the application range of the bio-ink is greatly widened, and the living cell co-culture scaffold can be constructed in vitro by using the bio-ink, so as to simulate the real environment of cells in vivo.

Preferably, the biocompatible organic component is hyaluronic acid and sodium alginate; the mass ratio of the hyaluronic acid to the sodium alginate is h: i is 5: (6-10), more preferably 5: 8.

preferably, the concentration of the inorganic component in the bio-ink is 0 to 2000 [ mu ] g/mL.

Preferably, the phosphate buffered saline solution is an existing product, such as Sangon Biotech (Shanghai) co., ltd. E607008-0500, which consists of: water, NaCl 136.89mM, KCl 2.67mM, Na₂HPO₄ 8.10mM、KH₂PO₄1.76mM、pH＝7.2～7.4。

Phosphate Buffered Saline (PBS) is a commonly used buffer solution for biological studies, and its osmolality and ion concentration are close to those of cells, so that it is used as a solvent for bio-ink to help maintain viability of cells. The PBS and the organic component must be formulated in the above ratio because the bio-ink must have a viscosity that maintains printing performance during printing and must not be too viscous to affect cell viability. Therefore, the proportion of the PBS and the organic component in the invention must be satisfied, and the inorganic component is added into a basic system formed under the condition that the proportion is satisfied, so that the printing performance and the functionality of the bio-ink can be realized, and the printing performance of the bio-ink can not be realized only by the PBS and the inorganic component.

In a fifth aspect, the present invention provides a method for preparing the above inorganic bio-ink capable of immunoregulation vascularization, comprising: preparing phosphate buffer salt solution; irradiating the bioactive inorganic component and the biocompatible organic component under ultraviolet light for sufficient sterilization; sequentially adding a biocompatible organic component and a bioactive inorganic component into a phosphate buffer solution to obtain a uniformly dispersed organic-inorganic mixture; and centrifuging the organic-inorganic mixture to remove air bubbles to finally obtain the biological ink capable of regulating the vascularization by immunity.

Preferably, the irradiation time under the ultraviolet lamp is 1 to 2 hours.

Preferably, taking biocompatible organic components of hyaluronic acid and sodium alginate as examples, the preparation method of the inorganic bio-ink comprises the following steps: preparing Phosphate Buffered Saline (PBS), Hyaluronic Acid (HA), Sodium Alginate (SA) and manganese silicate hollow nanospheres according to a certain proportion; placing the hyaluronic acid, the sodium alginate and the manganese silicate hollow nanospheres under ultraviolet light for irradiation and full sterilization; sequentially adding hyaluronic acid, manganese silicate hollow nanospheres and sodium alginate into phosphate buffer solution to obtain uniformly dispersed organic-inorganic mixture, and centrifuging the organic-inorganic mixture to remove bubbles to finally obtain the biological ink capable of regulating the vascularization.

Preferably, adding hyaluronic acid into phosphate buffer salt solution, shaking to fully dissolve, then adding the manganese silicate hollow nanospheres, performing ultrasonic treatment to uniformly disperse the manganese silicate hollow nanospheres, and finally adding sodium alginate and stirring uniformly; the mixing operation is carried out in a super clean bench.

In a sixth aspect, the present invention provides a bio-ink carrying living cells, comprising: the bio-ink and living cells dispersed in the bio-ink; the living cells are selected from at least one of immune cells and tissue cells, the immune cells are selected from at least one of macrophages, neutrophils and lymphocytes, and the tissue cells are selected from at least one of endothelial cells, fibroblasts and bone marrow mesenchymal stem cells. Preferably, the living cells have a cell density of 1X 10⁶～ 10×10⁶one/mL.

Preferably, when the living cells are tissue cells, the cell density is 4X 10⁶～6×10⁶Per mL; when the living cells are immune cells, the cell density is 4X 10⁶～6×10⁶one/mL.

In a seventh aspect, the invention provides a living cell co-culture scaffold, which is constructed by the above biological ink carrying living cells in vitro through biological 3D printing.

Preferably, the living cell co-culture scaffold comprises: a living cell co-culture scaffold carrying immune cells is used as an upper scaffold, and a living cell co-culture scaffold carrying tissue cells is used as a lower scaffold. Specifically, the living cell co-culture scaffold comprises the biological ink, tissue cells and immune cells; wherein the immune cells occupy the upper layer of the scaffold and the tissue cells occupy the lower layer of the scaffold.

In an eighth aspect, the present invention provides a method for preparing a living cell co-culture scaffold, comprising: preparing phosphate buffer salt solution; irradiating hyaluronic acid, sodium alginate and manganese silicate hollow nanospheres under ultraviolet light for sufficient sterilization; sequentially adding hyaluronic acid, manganese silicate hollow nanospheres, digested and centrifuged tissue cells or digested and centrifuged immune cells into phosphate buffer solution to obtain tissue cell suspension or immune cell suspension; respectively adding sodium alginate into the tissue cell suspension or the immune cell suspension, centrifuging to remove bubbles, and obtaining A biological ink containing tissue cells and B biological ink containing immune cells; and carrying out biological 3D printing to obtain the living cell co-culture scaffold.

Preferably, the cell density in the tissue cell suspension is (4-6) x 10⁶one/mL, preferably 5X 10⁶Per mL; the cell density in the immune cell suspension is (4-6) multiplied by 10⁶Per mL; preferably 5X 10⁶one/mL.

Preferably, adding hyaluronic acid into phosphate buffer salt solution, shaking to fully dissolve, then adding the manganese silicate hollow nanospheres, and performing ultrasonic treatment to uniformly disperse the manganese silicate hollow nanospheres to obtain mixed solution; digesting and centrifuging the tissue cells and the immune cells, respectively adding the tissue cells and the immune cells into the mixed solution, and uniformly blowing to obtain a tissue cell suspension and an immune cell suspension; finally, adding sodium alginate respectively; all the above operations are carried out in a sterile environment.

Preferably, the biometric 3D printing comprises: respectively loading biological ink A containing tissue cells and biological ink B containing immune cells into a needle cylinder of an extrusion biological 3D printer system, and setting a printing program; firstly, printing the biological ink A containing the histiocytes layer by layer, switching the needle cylinder after the required number of layers is reached, and continuously printing the biological ink B containing the immune cells layer by layer until the end, thus obtaining the scaffold for co-culturing the histiocytes and the immune cells.

Drawings

FIG. 1 is (a) a scanning electron micrograph of hollow nanospheres of manganese silicate prepared in the present invention obtained by a scanning electron microscope (Magellan400, FEI, USA), (b) a transmission electron micrograph obtained by a transmission electron microscope (JEM-2100F, Japan), (c) an X-ray diffraction analysis chart obtained by an X-ray diffractometer (D/max2550V, Rigaku, Japan), (D) N₂Adsorption-desorption curves obtained from a fully automatic four-station specific surface pore size analyzer (Quadrasorb SI, usa).

FIG. 2 is a distribution diagram and a combined diagram of Si, O and Mn elements of the hollow manganese silicate nanospheres prepared in the present invention, which are obtained by a transmission electron microscope (JEM-2100F, Japan) and an associated energy spectrometer (EDS).

Fig. 3 is a characteristic of degradation performance of the manganese silicate hollow nanospheres prepared in the present invention. Transmission electron microscope images of manganese silicate soaked in PBS at pH 7.4 (a) for 0h, (b) for 8h, and (c) for 36 h; (d) a Mn ion release curve, (e) a Si ion release curve; the ion release curve was measured by inductively coupled plasma emission spectroscopy (ICP-AES, Vista AX, USA).

FIG. 4 shows the cell proliferation of macrophages cultured with a series of manganese silicate hollow nanospheres at concentrations (0-500. mu.g/mL) for 1 day and 3 days.

FIG. 5 shows the expression of pro-inflammatory factors (a) TNF- α, (b) IL-6, (c) OSM, (d) IL-1 β, macrophage surface marker type M2 (e) CD206, anti-inflammatory factor (f) IL-10, (g) ARG-1, and vascular endothelial growth factor (h) VEGF after macrophage culture in hollow manganese silicate nanospheres of 0, 25, 100 μ g/mL.

FIG. 6 is a confocal image of DAPI-phalloidin (Phallodin) staining of the groups of FIG. 5 (0, 25, 100. mu.g/mL manganese silicate hollow nanospheres).

Fig. 7 shows the expression of VEGF pathway related receptor genes (a) VEGFR1, (b) VEGFR2, (c) PDGFR α, (d) PDGFR β and the typical angiogenetic gene (e) CD31 after endothelial cells are cultured in a conditioned medium, which is an immune microenvironment generated by the interaction of the manganese silicate hollow nanospheres with macrophages.

Fig. 8 is a macroscopic optical photograph and a microscopic photograph of (a) a viscosity-shear rate curve, (b) a modulus-angular frequency curve, and (c) a scaffold of the manganese silicate inorganic bio-ink prepared in the present invention.

FIG. 9 is a fluorescent staining pattern of viable cells of a macrophage (upper layer-green) and endothelial cell (lower layer-red) co-culture scaffold according to the present invention; (a) a side view, (b) a top view, and (c) a three-dimensional view.

FIG. 10 is a graph showing the survival of co-culture scaffolds prepared in the present invention. FIG. 10 (a) shows fluorescence staining images of live and dead cells (live cells-green, dead cells-red) after Co-0MS, Co-0.5MS, Co-1MS, Co-2MS coculture scaffolds were cultured for 0, 7, 14, 21 days; FIG. 10 (b) shows the cell viability statistics from left to right after Co-0MS, Co-0.5MS, Co-1MS, Co-2MS coculture scaffolds were cultured for 0, 7, 14, and 21 days.

Detailed Description

The present invention is further described below in conjunction with the following embodiments, which are intended to illustrate and not to limit the present invention.

Preparation of the manganese silicate hollow nanosphere:

ammonium fluoride (NH)₄F) And Cetyl Trimethyl Ammonium Bromide (CTAB) are dissolved in deionized water to form a uniform solution, Tetraethoxysilane (TEOS) is added dropwise after stirring for 1-2 h at 70-90 ℃, supernatant is collected after aging overnight at room temperature, and then centrifugation is carried out at 7000-9000 rpm for 12-16 minutes. And washing the obtained white product with deionized water and ethanol for 3-5 times respectively, and drying in vacuum at 50-70 ℃ to obtain the silicon dioxide nanospheres.

Dissolving the silicon dioxide nanospheres in deionized water and CH₃COONa and MnCl₂.4H₂Adding triethanolamine into the mixed solution of O until the pH value is 7.3-7.6, and adjusting the pH value to provide OH-, so as to obtain a mild alkalescent solution environment to promote the hydrolysis of the silicon dioxide into H₄SiO₄. Putting the obtained solution into a polytetrafluoroethylene reaction kettle and putting the reaction kettle into an oven, carrying out hydrothermal reaction for 22-26 hours at the temperature of 100-120 ℃, and hydrolyzing a small amount of silicon dioxide inside into H in the process₄SiO₄While generating active sites on the surface thereof to adsorb Mn²⁺. Subsequently, Mn²⁺And H₄SiO₄React with each other and deposit on the interface of the silica and the solution, forming small solid nanospheres on the surface of the silica, and finally, the same deposition and growth process continues to occur, forming complete hollow nanospheres stacked from nanospheres. And centrifuging the cooled reaction solution to obtain brown precipitate, and alternately washing the brown precipitate with water and absolute ethyl alcohol for 3-5 times. And calcining the washed particles in an electric furnace at 600-700 ℃ for 5-7 hours, and removing residual organic impurities to obtain the manganese silicate hollow nanospheres.

Preparation of biological ink:

(1) preparing materials: according to the required ink amount, the raw materials are prepared in the following proportion: 10mL of Phosphate Buffered Saline (PBS), 0.5g of Hyaluronic Acid (HA), 0.8g of Sodium Alginate (SA), and 0.005-0.02 g of manganese silicate hollow nanospheres.

(2) And (3) sterilization: (1) the hyaluronic acid, the sodium alginate and the manganese silicate hollow nanospheres are placed under ultraviolet lamp light to irradiate for 1 hour so as to achieve a sufficient sterilization effect.

(3) Mixing materials: adding hyaluronic acid into phosphate buffer solution, shaking to dissolve completely, adding manganese silicate hollow nanospheres, performing ultrasonic treatment to disperse the nanospheres uniformly, and finally adding sodium alginate and stirring uniformly. The above operations are all carried out in a clean bench.

(4) Removing bubbles: centrifuging the mixture obtained in the step (3) until all the mixture is deposited at the bottom of the container and no air bubbles exist, and obtaining the bio-ink.

Preparation of live cell co-culture scaffold:

preparing materials: according to the required ink quantity, the raw materials are prepared according to the following proportion: 10mL of Phosphate Buffered Saline (PBS), 0.5g of Hyaluronic Acid (HA), 0.8g of Sodium Alginate (SA), 0.005-0.02 g of manganese silicate hollow nanospheres, endothelial cells and macrophages.

And (3) sterilization: (1) the hyaluronic acid, the sodium alginate and the manganese silicate hollow nanospheres are placed under ultraviolet lamp light to irradiate for 1 hour so as to achieve a sufficient sterilization effect.

Mixing materials: adding hyaluronic acid into phosphate buffer solution, shaking to dissolve completely, adding manganese silicate hollow nanosphere, and ultrasonically dispersing to obtain endothelial cells and macrophage cellsDigesting and centrifuging the cells respectively, adding the cells into the mixed solution, and blowing and beating the cells uniformly to form cells with the density of 4-6 multiplied by 10⁶Cell suspension of preferably 5X 10 cells/mL⁶one/mL. And finally adding sodium alginate. The above operations are all carried out in a clean bench. The bio-ink containing endothelial cells is a bio-ink, and the bio-ink containing macrophages is referred to as B bio-ink.

Removing bubbles: bio-inks a and B were centrifuged until all settled at the bottom of the container and no air bubbles.

Printing: and respectively loading the biological ink A and the biological ink B into a needle cylinder of an extrusion type biological 3D printer system, and setting a printing program. Firstly, printing the biological ink A layer by layer, switching the needle cylinder after the required layers are reached, and continuously printing the biological ink B layer by layer above the ink A until the end to obtain the bracket for co-culturing the endothelial cells and the macrophages.

The present invention will be described in detail by way of examples. It is also to be understood that the following examples are illustrative of the present invention and are not to be construed as limiting the scope of the invention, and that certain insubstantial modifications and adaptations of the invention by those skilled in the art in light of the foregoing description are intended to be included within the scope of the invention. The specific process parameters and the like of the following examples are also only one example of suitable ranges, i.e., those skilled in the art can select the appropriate ranges through the description herein, and are not limited to the specific values exemplified below.

Example 1

Preparation of the manganese silicate hollow nanosphere:

(1) 3g of ammonium fluoride (NH)₄F) And 1.8g of cetyltrimethylammonium bromide (CTAB) in 500mL of deionized water to form a homogeneous solution, 9mL of Tetraethoxysilane (TEOS) was added dropwise after stirring at 80 ℃ for 1 hour, and after aging overnight at room temperature, the supernatant was collected and then centrifuged at 8000rpm for 15 minutes. And washing the obtained white product with deionized water and ethanol for 3 times respectively, and drying in vacuum at 60 ℃ to obtain the silicon dioxide nanospheres.

(2) 0.4g of silica nanospheres were dissolved in a solution containing 160mL of deionized water, 0.2g of CH₃COONa and 0.4g MnCl₂.4H₂O, then triethanolamine was added until the pH became 7.5. The resulting solution was divided into four equal portions, charged into four 50mL polytetrafluoroethylene reaction vessels and placed in an oven for hydrothermal reaction at 110 ℃ for 24 hours. The cooled reaction solution was centrifuged to obtain a brown precipitate, which was washed with water and anhydrous ethanol alternately 6 times. And calcining the washed particles in an electric furnace at 650 ℃ for 6 hours to remove organic impurities to obtain the manganese silicate nanospheres.

Fig. 1 and 2 are characterization results of manganese silicate nanospheres obtained according to the preparation method. Scanning electron microscope and transmission electron microscope images show that the synthesized manganese silicate is really a hollow structure and the diameter is about 100 nm. X-ray diffraction analysis shows that the phase is (Mn)₂O₃)₃MnSiO₃It is a brown manganese ore phase. The EDS surface scanning spectrogram of the elements shows that Mn, O and Si elements are uniformly distributed on the surface of the hollow sphere. The above characterization results confirm the successful preparation of the manganese silicate hollow nanospheres.

Figure 3 is a degradation performance characterization of the manganese silicate hollow nanospheres. The transmission electron microscope images show that the manganese silicate keeps a hollow structure with uniform size and complete structure before any treatment, and after the manganese silicate nanospheres are immersed in a PBS solution (pH 7.4) for 8 hours, the structure of the manganese silicate nanospheres is damaged, but the hollow structure is still visible, and the hollow structure tends to be blurred and flaky fragments appear after the manganese silicate nanospheres are continuously immersed for 36 hours. The ion release results show that Mn ions and Si ions released from manganese silicate rapidly rise in the first 12 hours and then are gradually and slowly released, and the Mn ions and Si ions in the solution reach concentrations of 5.85 mug/mL and 105.44 mug/mL at 72 hours.

Example 2

The immune regulation of the manganese silicate hollow nanosphere has the vascular function:

(1) mixing macrophage with 10⁴Initial density of individual/well was seeded in 96-well plates. After 2 hours, the medium was replaced with 100 μ L of modified medium containing gradient concentration hollow nanospheres of manganese silicate (0, 5, 10, 25, 50, 100, 250, 500 μ g/mL) formulated as ready-made products with manganese silicate prepared according to the present invention and continued. Preparation method of 0 mu g/mL culture medium: DMEM high-glucose medium (Thermo Fisher Scientific, Grand Island, American) + 10% (v/v) fetal bovine serum (Thermo Fisher Scientific, FBS, Gibco) + 1% (v/v) penicillin/streptomycin (Thermo Fisher Scientific). Weighing the hollow manganese silicate nanospheres with corresponding weight, adding the hollow manganese silicate nanospheres into the culture medium, and performing ultrasonic dispersion to form uniform suspension, thereby obtaining the modified culture medium containing the hollow manganese silicate nanospheres. After 3 days of culture, 200. mu.L of CCK-8 assay was added to each well by removing the old medium, and after 1 hour of incubation, 100. mu.L of the assay was pipetted into a new 96-well plate. The absorbance of the solution at 450nm was recorded using a microplate reader, and the results are shown in FIG. 4. 250. 500 mu g/mL shows obvious inhibition effect on macrophage proliferation, which indicates that the concentration range has toxic and side effect. 5. 10, 100. mu.g/mL did not differ significantly from the control group. 25. The proliferation of the cells was significantly promoted by 50. mu.g/mL compared to the control group. The manganese silicate modified medium of 100 mu g/mL is the maximum concentration which does not affect the proliferation of macrophages, the modified medium of 25 mu g/mL shows the best proliferation promoting effect, and the two concentrations are preferably used as experimental groups for subsequent RT-qPCR experiments.

(2) Subjecting macrophages to conventional culture medium 10⁵Initial density of individual/well was seeded in 6-well plates. After 24h, the medium was changed to 2mL of modified medium containing different concentrations of manganese silicate hollow nanospheres (0, 25, 100. mu.g/mL) for 3 days. Total RNA is extracted from cells by using Trizol reagent, then the expression of related genes in macrophages is detected by using RT-qPCR technology, and simultaneously the morphology of each group of cells is photographed by using a confocal microscope.

(3) Collecting the culture medium supernatant after culturing the macrophages in the step (2) to obtain an immune microenvironment in which the manganese silicate and the macrophages interact, and culturing the endothelial cells by taking the immune microenvironment as a conditioned medium. Endothelial cells were cultured at 5X 10⁴Initial density of individual/well was seeded in 6-well plates. After 24 hours, the medium was replaced with 2mL of conditioned medium, and after further culturing for 3 days, endothelial cell RNA was extracted and the expression of the angiogenesis-associated gene was detected in the same manner as in (2).

FIG. 5 shows that after macrophage cells are cultured by manganese silicate with concentration of 25 and 100 mug/mL for 3 days, expression of proinflammatory factors TNF-alpha, IL-6, OSM and IL-1 beta is down-regulated, expression of anti-inflammatory factor IL-10 and ARG-1 is up-regulated, and expression of macrophage cell surface marker CD206 of M2 type is up-regulated, compared with a control group without the manganese silicate hollow nanospheres, and the macrophage cells cultured by manganese silicate are proved to generate polarization, namely phenotype transformation under the stimulation of bioactive ions, and the manganese silicate hollow nanospheres can effectively promote the macrophage cells to enter an inflammation inhibiting state and show an M2 phenotype. At the same time, the expression of the vascular endothelial growth factor VEGF is relatively up-regulated. The gene expression of macrophages cultured with 100. mu.g/mL manganese silicate is more significant than that of the 25. mu.g/mL group (see FIGS. 5a-h), indicating that the macrophages have stronger immunoregulatory function.

The different phenotypes of macrophages have their own morphology. Compared to the M1 phenotype, M2 macrophages are more irregular and develop many small filopodia. The morphology of macrophages cultured at the above specified manganese silicate concentration was observed by confocal laser scanning microscopy. Phalloidin staining of F-actin revealed the cytoskeletal organization of the cells (see fig. 6). Macrophages cultured in conventional media mostly approached a round shape and were not stained significantly for phalloidin, whereas macrophages cultured with 25 μ g/mL and 100 μ g/mL manganese silicates were irregular in shape and strongly stained by phalloidin, indicating that manganese silicate is effective in promoting actin polymerization and that macrophages switch to the M2 phenotype in response to manganese silicate. Combining gene expression and cell morphology results, the synthesized manganese silicate hollow nanosphere successfully stimulates macrophages to achieve an M2 anti-inflammatory phenotype.

The gene expression result of the immune microenvironment generated by the interaction of the manganese silicate hollow nanospheres and the macrophages after 3 days of culture of endothelial cells in conditioned medium is shown in figure 7. The expression of a typical angiogenesis-related gene CD31 is up-regulated, and the expressions of VEGF pathway-related receptor genes VEGFR1, VEGFR2, PDGFR alpha and PDGFR beta are also up-regulated, so that the immune microenvironment activates the VEGF-related pathways in endothelial cells, the VEGF-related pathways have a good effect of promoting angiogenesis in vitro at a cell level, and preferably, a 100 mu g/mL conditioned medium has a more remarkable effect on the gene up-regulation and shows concentration dependence.

Example 3

Preparing and printing the manganese silicate inorganic biological ink:

(1) preparing materials: 40mL of Phosphate Buffered Saline (PBS) is equally divided into 4 parts, 2g of Hyaluronic Acid (HA) is equally divided into 4 parts, 3.2g of Sodium Alginate (SA) is equally divided into 4 parts, and 0g, 0.005g, 0.01g and 0.02g of manganese silicate hollow nanospheres are respectively added.

(3) Mixing materials: 0.5g of hyaluronic acid was added to 10mL of Phosphate Buffered Saline (PBS), and shaken to be sufficiently dissolved, to obtain 4 groups of hyaluronic acid solutions. Then respectively adding 0/0.005g/0.01g/0.02g of manganese silicate hollow nanospheres, performing ultrasonic treatment to uniformly disperse the manganese silicate hollow nanospheres, finally adding sodium alginate, and uniformly stirring to obtain four groups of biological inks, wherein the four groups of biological inks are respectively named as 0MS, 0.5MS, 1MS and 2MS (the concentrations of the manganese silicate hollow nanospheres are respectively 0 mug/mL, 500 mug/mL, 1000 mug/mL and 2000 mug/mL). The above operations are all carried out in a clean bench.

(4) Removing bubbles: centrifuging the mixture obtained in (3) until all is deposited at the bottom of the container and no air bubbles are formed.

Printing: adding the biological ink into a needle cylinder of the extrusion type biological 3D printer system, and transferring the biological ink to an air pressure pushing extrusion type printing module. The printing procedure was set for a square porous scaffold with a side length of 1cm, a root spacing of 1.2mm, a single layer height of 0.08mm, and a number of layers of 20. Each printed layer is converted by 90 °, and the pore structure is also square. The inorganic biological ink is extruded by pushing an extrusion type needle cylinder through air pressure, the pressure range is 210-260 kPa, and the obtained supports correspond to the ink and are named as 0MS, 0.5MS, 1MS and 2MS supports.

Characterization of each of the above inorganic bio-inks and scaffolds (see fig. 8) shows that each group has shear thinning properties and high viscosity after release of shear stress, which facilitates extrusion of the ink from a fine nozzle to form a fixed size filament. For the same component, the storage modulus of the biological ink is larger than the loss modulus, the material is in a gel state, the biological ink is used for printing, and the support has excellent printing performance and formability, so that the biological ink is very suitable for 3D printing.

Example 4

The manganese silicate inorganic biological ink is used for preparing a living cell co-culture scaffold and the biological activity thereof:

(1) preparing materials: 40mL Phosphate Buffered Saline (PBS) is equally divided into 4 parts, 2g Hyaluronic Acid (HA) is equally divided into 4 parts, 3.2g Sodium Alginate (SA) is equally divided into 4 parts, 0g, 0.005g, 0.01g, 0.02g manganese silicate hollow nanosphere and macrophage are respectively added into the solution, and the solution is about 2 multiplied by 10⁹Endothelial cells of about 2X 10⁹And (4) respectively.

(3) Mixing materials: 0.5g of hyaluronic acid was added to 10mL of Phosphate Buffered Saline (PBS), and shaken to be sufficiently dissolved, to obtain 4 groups of hyaluronic acid solutions. Then respectively adding 0/0.005/0.01/0.02g of manganese silicate hollow nanospheres, performing ultrasonic treatment to uniformly disperse the nanospheres, dividing the mixed solution into two parts, respectively adding macrophages and endothelial cells to make the density of the mixed solution be 5 multiplied by 10⁶Finally, sodium alginate is added into each/mL of the mixture and stirred uniformly to obtain four groups of biological ink with cells, which are named as Co-0MS, Co-0.5MS, Co-1MS and Co-2MS respectively, wherein each group of ink is divided into two types, and the operations are carried out in a superclean bench respectively.

(5) Printing: and respectively adding each group of biological ink into the needle cylinders with different serial numbers of the extrusion type biological 3D printer system, and transferring to an extrusion type printing module. The printing procedure was set for a square, porous scaffold with 1cm side length, 1.2mm root spacing, 0.08mm single layer height, and 20 layers (10 layers of endothelial cells below, 10 layers of macrophages above). Each printed layer is converted by 90 °, and the pore structure is also square. The inorganic biological ink is extruded by pushing the extrusion type syringe by air pressure, the pressure range is 210-260 kPa, and the obtained supports correspond to the ink and are named as 0MS, 0.5MS, 1MS and 2MS supports.

(6) Live and dead staining: the above live cell co-culture scaffolds were cultured to specific time points (0, 7, 14, 21 days) and then live-dead staining was performed. The live-dead staining solution was prepared in a ratio of 1mL to 2 μ L to 3 μ L of PBS, Calcein-AM PI, 2mL of the live-dead staining solution was added to each scaffold, the scaffold was incubated in an incubator at 37 ℃ for 10 minutes, washed with PBS, and then photographed using a fluorescence microscope. Green fluorescence corresponds to live cells and red fluorescence to dead cells.

The above-described printed cell co-culture scaffold had a cell distribution consistent with the model design, with macrophages occupying the upper layer of the scaffold and endothelial cells occupying the lower layer of the scaffold (see fig. 9). The biological 3D printing strategy can realize accurate arrangement of cell space, so that a required immune microenvironment is constructed. Cell survival and death staining patterns and semi-quantitative statistical results prove that the cell survival rate in the co-culture scaffold can still be maintained above 90% after three weeks (see fig. 10), and the effect is not good or bad among all groups, which indicates that the printing property and the cell activity can be satisfied within the concentration range, and proves that the bio-ink has good biocompatibility and the feasibility of constructing the living cell co-culture scaffold by the method.

Claims

1. The manganese silicate hollow nanosphere is characterized in that the manganese silicate hollow nanosphere is a brown manganese ore phase, has a hollow structure and has a chemical composition of (Mn)₂O₃)₃MnSiO₃(ii) a Preferably, the diameter of the manganese silicate hollow nanosphere is 90-100 nm, and the specific surface area is 630-640 m²/g。

2. A method for preparing hollow nanospheres of manganese silicate as claimed in claim 1 wherein silica nanospheres are dissolved in a solution comprising deionized water, CH₃COONa or Na₂SO₄And MnCl₂.4H₂Adding triethanolamine or ethanolamine into the mixed solution of O until the pH value is 7.3-7.6; and then carrying out hydrothermal reaction at 100-120 ℃ for 22-26 hours, and finally calcining at 600-700 ℃ for 5-7 hours to obtain the manganese silicate hollow nanospheres.

3. The method of claim 2, wherein the step of preparing the composition is carried out in a batch processThe particle size of the silicon dioxide nanospheres is 100-120 nm; the silica nanosphere and CH₃The mass ratio of COONa is (1-3): 1; the silicon dioxide nanospheres and MnCl₂.4H₂The mass ratio of O is (0.5-2): 1.

4. a modified culture medium, comprising: a basal medium, and the manganese silicate hollow nanospheres of claim 1 dispersed in the basal medium;

the basic culture medium comprises the following components: the basic culture medium comprises the following components: DMEM high-sugar medium, fetal calf serum, penicillin/streptomycin;

the concentration of the manganese silicate hollow nanosphere is below 100 mug/mL.

5. The modified culture medium of claim 4, wherein the concentration of the manganese silicate hollow nanospheres is 25-100 μ g/mL.

6. A bio-ink, comprising a phosphate buffer solution, and a bioactive inorganic component and a biocompatible organic component dispersed in the phosphate buffer solution; the biological active inorganic component is the manganese silicate hollow nanosphere of claim 1, and the biological compatible organic component is at least two of sodium alginate, hyaluronic acid, collagen, methylcellulose and carrageenan;

the mass ratio of the bioactive inorganic component to the biocompatible organic component is (0-0.2): 13.

7. the bio-ink according to claim 6, wherein the biocompatible organic component is hyaluronic acid and sodium alginate; the mass ratio of the hyaluronic acid to the sodium alginate is h: i = 5: (6-10), preferably 5: 8.

8. the bio-ink according to claim 6 or 7, wherein the concentration of the inorganic component in the bio-ink is 0 to 2000 μ g/mL.

9. A method of making the bio-ink of any one of claims 6 to 8, comprising:

preparing phosphate buffer salt solution; irradiating the bioactive inorganic component and the biocompatible organic component under ultraviolet light for sufficient sterilization;

sequentially adding a biocompatible organic component and a bioactive inorganic component into a phosphate buffer solution to obtain a uniformly dispersed organic-inorganic mixture;

and centrifuging the organic-inorganic mixture to remove air bubbles to finally obtain the biological ink capable of regulating the vascularization by immunity.

10. The method of claim 9, wherein the irradiation time under the ultraviolet lamp is 1 to 2 hours.

11. The preparation method according to claim 9 or 10, wherein when the biocompatible organic component is hyaluronic acid and sodium alginate, hyaluronic acid, manganese silicate hollow nanospheres and sodium alginate are sequentially added to a phosphate buffered saline solution to obtain a uniformly dispersed organic-inorganic mixture; preferably, adding hyaluronic acid into phosphate buffer salt solution, shaking to fully dissolve, then adding the manganese silicate hollow nanospheres, performing ultrasonic treatment to uniformly disperse the manganese silicate hollow nanospheres, and finally adding sodium alginate and stirring uniformly; the mixing operation is carried out in a super clean bench.

12. A bio-ink carrying living cells, comprising: the bio-ink of claims 6-8, and living cells dispersed in the bio-ink; the living cells are selected from at least one of immune cells and tissue cells, the immune cells are selected from at least one of macrophages, neutrophils and lymphocytes, and the tissue cells are selected from at least one of endothelial cells, fibroblasts and bone marrow mesenchymal stem cells; preferably, the viable cells have a cell density of1×10⁶～10×10⁶one/mL.

13. The bio-ink containing living cells according to claim 12, wherein when the living cells are tissue cells, the cell density is 4 x 10⁶～6×10⁶Per mL; when the living cells are immune cells, the cell density is 4X 10⁶～6×10⁶one/mL.

14. A living cell co-culture scaffold, which is constructed in vitro from the living cell-loaded bio-ink according to claim 12 or 13 by bio-3D printing.

15. The living cell co-culture scaffold according to claim 14, comprising: a living cell co-culture scaffold carrying immune cells is used as an upper scaffold, and a living cell co-culture scaffold carrying tissue cells is used as a lower scaffold.

16. A method for preparing a scaffold for co-culture of living cells according to claim 14 or 15, comprising:

preparing phosphate buffer salt solution;

irradiating hyaluronic acid, sodium alginate and manganese silicate hollow nanospheres under ultraviolet light for sufficient sterilization;

sequentially adding hyaluronic acid, manganese silicate hollow nanospheres, digested and centrifuged tissue cells or digested and centrifuged immune cells into phosphate buffer solution to obtain tissue cell suspension or immune cell suspension;

respectively adding sodium alginate into the tissue cell suspension or the immune cell suspension, centrifuging to remove bubbles, and obtaining A biological ink containing tissue cells and B biological ink containing immune cells;

and carrying out biological 3D printing to obtain the living cell co-culture scaffold.

17. The method according to claim 16, wherein the cell density of the tissue cell suspension is (4-6) x 10⁶Per mL; the cell density in the immune cell suspension is (4-6) multiplied by 10⁶one/mL.

18. The preparation method according to claim 16 or 17, wherein hyaluronic acid is added to phosphate buffered saline solution, shaken to be sufficiently dissolved, and then manganese silicate hollow nanospheres are added and uniformly dispersed by ultrasound to obtain a mixed solution; digesting and centrifuging the tissue cells and the immune cells, respectively adding the tissue cells and the immune cells into the mixed solution, and uniformly blowing to obtain a tissue cell suspension and an immune cell suspension; finally, adding sodium alginate respectively; all the above operations are carried out in a sterile environment.

19. The method of any one of claims 16-18, wherein the biological 3D printing comprises:

respectively loading biological ink A containing tissue cells and biological ink B containing immune cells into a needle cylinder of an extrusion biological 3D printer system, and setting a printing program;

firstly, printing the biological ink A containing the histiocytes layer by layer, switching the needle cylinder after the required number of layers is reached, and continuously printing the biological ink B containing the immune cells layer by layer until the end, thus obtaining the scaffold for co-culturing the histiocytes and the immune cells.