CN114870071B

CN114870071B - Silicon-based bioactive ink, natural inorganic silicon-based material flexible three-dimensional porous support and application

Info

Publication number: CN114870071B
Application number: CN202210488949.5A
Authority: CN
Inventors: 吴成铁; 马景阁
Original assignee: Shanghai Institute of Ceramics of CAS
Current assignee: Shanghai Institute of Ceramics of CAS
Priority date: 2022-04-29
Filing date: 2022-04-29
Publication date: 2023-08-04
Anticipated expiration: 2042-04-29
Also published as: CN114870071A

Abstract

The invention discloses a flexible three-dimensional porous support made of silicon-based bioactive ink and natural inorganic silicon-based materials and application thereof. The silicon-based bioactive ink comprises a gel matrix and hard silicon-based mineral micro-nano particles embedded in the gel matrix, wherein the mass of the hard silicon-based mineral micro-nano particles is less than 30% of the gel matrix, and preferably is 1-20%. The flexible three-dimensional porous scaffold made of the natural inorganic silicon-based material has a regular porous structure, and the internal hard silicon-based mineral micro-nano particles can be slowly degraded and release bioactive ions to promote angiogenesis so as to accelerate skin repair, so that the flexible three-dimensional porous scaffold has important significance for treating severe burn and scald wound surfaces.

Description

Silicon-based bioactive ink, natural inorganic silicon-based material flexible three-dimensional porous support and application

Technical Field

The invention belongs to the field of bioengineering materials, and particularly relates to a flexible three-dimensional porous support made of silicon-based bioactive ink and natural inorganic silicon-based materials and application thereof.

Background

Skin is the first barrier to protect the environment in the human body, and plays an important role in maintaining body temperature, preventing water loss, regulating metabolic processes, resisting external invasion and the like. High temperature, chemical corrosion, electric shock, etc. cause severe skin burn. Severe burns can lead to disruption of the vascular network and dermal matrix within the skin tissue, in which case the difficulty of skin healing increases significantly and may deteriorate into chronic wounds. Therefore, there is a need to develop a multifunctional wound dressing capable of supporting gas exchange, retaining moisture of the wound surface and promoting revascularization and collagen deposition to accelerate skin tissue regeneration of burn wound surfaces.

The 3D printing technology is used as an additive manufacturing technology for layer-by-layer deposition, and can realize accurate regulation and control of distribution of biological materials in a three-dimensional space. The three-dimensional scaffold prepared by the extrusion type 3D printing technology can promote gas exchange, cell migration and growth of surrounding tissues. Thus, 3D printed three-dimensional scaffolds have great potential to be used as wound dressings.

Disclosure of Invention

Aiming at the problems, the invention provides the silicon-based bioactive ink, the natural inorganic silicon-based flexible three-dimensional porous scaffold and the application thereof, wherein the natural inorganic silicon-based flexible three-dimensional porous scaffold has a regular porous structure, and the internal hard (natural) silicon-based mineral micro-nano particles can be slowly degraded and release bioactive ions to promote angiogenesis so as to accelerate skin repair, so that the natural inorganic silicon-based flexible three-dimensional porous scaffold has important significance for treating severe burn and scald wound surfaces.

In a first aspect, the present invention provides a silicon-based bioactive ink. The silicon-based bioactive ink comprises a gel matrix and hard silicon-based mineral micro-nano particles embedded in the gel matrix, wherein the mass of the hard silicon-based mineral micro-nano particles is less than 30% of the gel matrix, and preferably is 1-20%. The mass of the hard silicon-based mineral micro-nano particles is within the range, and the bioactive ions generated by the stable degradation of the hard silicon-based mineral micro-nano particles in the silicon-based bioactive ink can positively influence the vital activities of skin cells, so that a bioactive ion microenvironment with proper concentration is established, and the migration and proliferation of cells and the expression of related genes are promoted.

Preferably, the hard silicon-based mineral micro-nano particles are diatomite micro-nano particles.

Preferably, the particle size of the hard silicon-based mineral micro-nano particles is less than 20 μm, preferably 5-15 μm.

Preferably, the gel matrix comprises one or more of hyaluronic acid gel, methacryloylated gelatin and sodium alginate gel; preferably, the gel matrix is a methacryloylated gelatin.

Preferably, the mass of the hard silicon-based mineral micro-nano particles is 5-20% of that of the gel matrix.

In a second aspect, the present invention provides a method for preparing a silicon-based bioactive ink as described in any one of the above. Dispersing the silicon-based mineral micro-nano particles in a solvent to form silicon-based mineral micro-nano particle dispersion liquid with the mass fraction of the silicon-based mineral micro-nano particles of 0.1-3.0%; dispersing a gel matrix or the gel matrix and a photoinitiator in a solvent to form a gel solution with the mass fraction of the gel matrix of 12-16%; uniformly mixing the hard silicon-based mineral micro-nano particle dispersion liquid and the gel solution to obtain the silicon-based bioactive ink; preferably, the volume ratio of dispersion to gel solution is 1:1.

Preferably, the solvent is independently selected from at least one of deionized water, ultrapure water, and phosphate buffer solution.

In a third aspect, the present invention provides a flexible three-dimensional porous scaffold of natural inorganic silicon-based material (natural inorganic silicon-based material composite three-dimensional scaffold). The flexible three-dimensional porous scaffold made of the natural inorganic silicon-based material has a regular porous structure, and comprises a basic framework formed by solidifying a gel matrix and hard silicon-based mineral micro-nano particles embedded into the framework and used as active factors for regulating cell behaviors, wherein the mass of the hard silicon-based mineral micro-nano particles is less than 30% of the gel matrix, and preferably is 1-20%. Preferably, the hard silicon-based mineral micro-nano particles are diatomite micro-nano particles.

Preferably, the natural inorganic silicon-based flexible three-dimensional porous scaffold is formed by 3D printing and then crosslinking and curing of silicon-based bioactive ink, and the silicon-based bioactive ink comprises a gel matrix and hard silicon-based mineral micro-nano particles embedded in the gel matrix. The mass of the hard silicon-based mineral micro-nano particles is less than 30% of that of the gel matrix, and is preferably 1-20%.

In a fourth aspect, the invention provides the use of a flexible three-dimensional porous scaffold of any one of the above-mentioned silicon-based bioactive inks or any one of the above-mentioned natural inorganic silicon-based materials in bioengineering, in particular for the treatment of skin scalds, burns and wounds.

The invention provides the preparation of the natural inorganic silicon-based material composite three-dimensional scaffold by using diatomite micro-nano particles for the first time, so that the embedding of the natural silicon-based mineral micro-nano particles in the hydrogel three-dimensional porous scaffold is realized. The composition endows the scaffold with higher mechanical property and biological activity, obviously improves the adhesion and spreading of skin cells on the surface of the scaffold, and realizes the construction of the tissue engineering scaffold with cost-effective and vascularization promoting activity.

Drawings

Fig. 1 is a (a, b) scanning electron micrograph and (c) X-ray diffraction analysis chart of diatomaceous earth microparticles.

FIG. 2 is a graph showing the cytocompatibility characterization of microparticles of diatomaceous earth, including proliferation of (a) fibroblasts and (b) vascular endothelial cells, within 5 days of culture in a diatomaceous earth dispersion medium. The bar graph of FIG. 2 is CTR, 10 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL, 250 μg/mL, 500 μg/mL, in that order from left to right.

Fig. 3 is a rheological characterization of the diatomite composite ink with different diatomite contents, showing that (a) the methacryloylated gelatin ink has temperature sensitivity and (b) the diatomite composite ink has temperature sensitivity and (c) both the methacryloylated gelatin ink and the diatomite composite ink have shear thinning properties. (c) The shear thinning properties of the five inks of (a) are quite similar and overlap is unavoidable, which does not affect the disclosure of the invention.

FIG. 4 is a representation of a 3D printed diatomite composite three-dimensional stent (Gel, 5DE-Gel, 10DE-Gel, 20DE-Gel, 30 DE-Gel), including (a) an appearance photograph, (b) an internal scanning electron microscope photograph, and (c) a Si, O element distribution photograph of the stent. The scales of (b) are all 100. Mu.m.

FIG. 5 shows (a, b) a fluorescence micrograph of a cell distribution on days 1 and 5 of a diatomite composite three-dimensional scaffold inoculated with fibroblasts and (c) proliferation of fibroblasts on the scaffold within 5 days. The scales of (a) and (b) are each 500. Mu.m. (c) Is sequentially Gel, 5DE-Gel, 10DE-Gel, 20DE-Gel, 30DE-Gel from left to right.

FIG. 6 shows (a, b) a fluorescent micrograph of a cell distribution on days 1 and 5 of a diatomite composite three-dimensional stent inoculated with vascular endothelial cells, statistics of (c) number and (d) cell area of vascular endothelial cells on the stent on day 1, and proliferation of vascular endothelial cells on the stent within 5 days (e). The scales of (a) and (b) are each 500. Mu.m. (e) Is sequentially Gel, 5DE-Gel, 10DE-Gel, 20DE-Gel, 30DE-Gel from left to right.

FIG. 7 shows the Si ion release profile of (a) angiogenesis-related gene expression and (b) scaffolds of a diatomite-composite three-dimensional scaffold seeded with vascular endothelial cells. (a) Is sequentially Gel, 5DE-Gel, 10DE-Gel, 20DE-Gel from left to right. (b) Sequentially from bottom to top, refers to Gel, 5DE-Gel, 10DE-Gel, 20DE-Gel, 30DE-Gel.

FIG. 8 is a study of a 5DE-Gel diatomite composite three-dimensional stent for wound treatment of skin scalds, including (a) photographs of the wound and (b) statistical results of relative wound areas over 14 days; histological staining analysis included (c) Masson trichromatic staining (collagen fibers: blue) and (d) collagen fiber content statistics, (e) CD31 immunofluorescent staining (blood vessels: green, nuclei: blue) and (f) blood vessel number statistics. (b) From left to right, are Blank, gel, 5DE-Gel in that order. (a) Is 1cm, (c) is 500 μm and (e) is 200 μm.

Detailed Description

The invention is further illustrated by the following embodiments, which are to be understood as merely illustrative of the invention and not limiting thereof. Unless otherwise specified, each percentage refers to a mass percent.

The present disclosure provides a silicon-based bioactive ink. The silicon-based bioactive ink comprises a gel matrix and hard (natural) silicon-based mineral micro-nano particles uniformly embedded in the gel matrix. The mass of the hard silicon-based mineral micro-nano particles is 1-20% of that of the gel matrix. If the mass ratio of the hard silicon-based mineral micro-nano particles is lower than 1%, the content of the silicon-based mineral micro-nano particles is too low, the improvement effect on the biological performance of the ink and the bracket is not obvious, the concentration of the released bioactive ions is not in the effective range, and the regulation and control on the cell behaviors are difficult to realize. If the mass ratio of the hard silicon-based mineral micro-nano particles is higher than 20%, the crosslinking of a gel matrix polymer network is influenced, the structural stability and mechanical performance of the scaffold are poor, and meanwhile, the high concentration of ions released by the excessively high silicon-based mineral micro-nano particles can inhibit the activity of cells and reduce the survival rate of the cells.

The hard silicon-based mineral micro-nano particles are bioactive inorganic materials derived from natural silicon-based minerals, and are micro-or nano-particles capable of keeping stable release of bioactive ions in a physiological environment. The bioactive ion comprises one or more of Ca, si, mg, zn ions. For example, the bioactive ion is a Si ion.

The hard silicon-based mineral micro-nano particles may be porous natural nonmetallic minerals. In some technical schemes, the natural silicon-based mineral micro-nano particles are natural diatomite micro-nano particles. The diatomite is a skeleton deposited by natural unicellular aquatic plant diatom, and the main component of the diatomite is SiO ₂ ·nH ₂ O. The diatomite particles have a highly regular nano-scale porous structure and have the characteristics of good mechanical strength, excellent absorption performance, high specific surface area, high hydrophilicity and the like. Compared with the silicon dioxide synthesized by a chemical method, the diatomite has no problems of complicated steps and impurity introduction, is a potential substitute for artificially synthesized silicon dioxide, and can be used as an inorganic 'growth factor' for improving the performance of bioactive ink and regulating and controlling the vital activity of cells.

The invention provides the bioactive ink containing natural diatomite for the first time. The diatomite micro-nano particles are uniformly dispersed in the ink and release bioactive ions, and the obtained ink has good printability and formability and can be cured and crosslinked under blue light irradiation. Although natural silicon-based minerals are various, diatomaceous earth is selected for use in the present invention because it can provide performance advantages not provided by other silicon-based minerals. Diatomite is a non-metal mineral of biological origin, has near neutral pH value, is nontoxic, is insoluble in most acids, and has good biocompatibility and stability. In addition, the diatomite micro-nano particles are distributed with highly regular and dense nano-pore structures compared with other silicon-based minerals such as silicate minerals, which gives the diatomite strong adsorption performance. The excellent water absorption performance enables the natural diatomite composite ink and the bracket to have high water content and water retention, which is the performance of the skin burn wound dressing, can keep a relatively moist environment for the wound surface, and is beneficial to wound healing. Meanwhile, a large number of hydroxyl groups on the surface of the natural diatomite can also enhance the adsorption capacity of the ink and the bracket to protein, and the natural diatomite is also beneficial to promoting the skin repair process. This is manifested in that diatomaceous earth can increase the surface roughness of the ink, providing effective binding sites for cell adhesion and migration.

The particle size of the hard silicon-based mineral micro-nano particles is smaller than 20 mu m. The purpose of controlling the particle size of the fine nano particles of the silicon-based mineral in the above range is to ensure that the addition of the fine nano particles of the silicon-based mineral does not affect the printability of the ink and the gel matrix forming properties, such as agglomeration and clogging during extrusion printing. In some technical schemes, the hard silicon-based mineral micro-nano particles are screened by a dry screening method so that the particle size of the particles is smaller than 20 mu m. Preferably, the particle size of the hard silicon-based mineral micro-nano particles is 5-15 μm.

The gel matrix comprises one or more of hyaluronic acid gel, methacryloylated gelatin and sodium alginate gel. The gel matrix has good biocompatibility. Preferably, the gel matrix is methacryloylated gelatin (GelMA). The methacryloylated gelatin has good biocompatibility, and degradation products thereof have no cytotoxicity.

In some technical schemes, the mass of the hard diatomite micro-nano particles is 5-20% of that of the methacryloylated gelatin. Therefore, the printability and photocrosslinkability of the ink can be further improved, and the problem that the concentration of released ions is high and the cell survival is not favored is avoided.

The invention also provides a preparation method of the silicon-based bioactive ink. Dispersing the silicon-based mineral micro-nano particles in a solvent to form a silicon-based mineral micro-nano particle dispersion liquid with the mass fraction of the silicon-based mineral micro-nano particles of 0.1-3.0% (preferably 0.12-2.4%); dispersing the gel matrix (and the photoinitiator) in a solvent to form a gel solution with the mass fraction of the gel matrix being 12-16%; uniformly mixing the dispersion liquid and the gel solution of the hard silicon-based mineral micro-nano particles to obtain the silicon-based bioactive ink.

In some technical schemes, diatomite powder is soaked in a solvent, and diatomite dispersion liquid is prepared through ultrasonic dispersion. The methacryloylated gelatin (GelMA) and the photoinitiator were dissolved in a solvent at 65℃to obtain a GelMA hydrogel solution. The diatomite dispersion liquid and the GelMA hydrogel solution are fully mixed and then cooled at the temperature of 4 ℃ to form the printable silicon-based bioactive ink. Before the diatomite is used, the diatomite from the diatom deposition exoskeleton is subjected to particle size separation by a dry screening method, specifically, the diatomite powder in a dry state is screened by a 500-mesh screen, and the screened particles are sterilized under ultraviolet light for more than 1 hour and are used for preparing the bioactive ink.

The present disclosure also provides a natural inorganic silicon-based material composite three-dimensional scaffold (natural inorganic silicon-based material flexible three-dimensional porous scaffold) having a regular three-dimensional porous structure. Namely, the invention applies the hard natural silicon-based mineral micro-nano particles to be used for the flexible three-dimensional porous scaffold for the first time. The composite three-dimensional scaffold comprises a basic framework formed by solidifying a gel matrix and hard silicon-based mineral micro-nano particles which are embedded into the framework and used as active factors for regulating and controlling cell behaviors. The diatomite has light weight, low density, high hydrophilicity and high adsorptivity, mainly comes from the unique regular nano porous structure and the surface distributed silicon hydroxyl groups which are naturally formed by the diatomite, can enhance the mechanical property, the hydrophilic property and the surface roughness of the bracket, endows the bracket with good water absorbability and protein adsorptivity, and does not influence the pH value and the biocompatibility of the bracket. The scaffold is characterized in that natural silicon-based mineral micro-nano particles embedded in the scaffold are used as 'active factors' capable of improving the scaffold performance and regulating cell behaviors, on one hand, the mechanical performance, the hydrophilic performance and the surface roughness of the scaffold are enhanced, and on the other hand, bioactive ions released through degradation are used for stimulating cell spreading, migration, proliferation and differentiation. The stent can be used as a burn/scald wound dressing, accelerates skin repair by promoting blood vessel regeneration, and has great application potential in the aspect of treating severe burns and scald skin wounds.

As an example, the natural inorganic silicon-based material composite three-dimensional scaffold is integrally constructed by bioactive ink compounded by natural silicon-based mineral micro-nano particles and gel matrix gel, and has a regular porous structure. In the culturing process, the natural silicon-based mineral micro-nano particles in the stent slowly degrade to release Si ions with proper concentration, and the natural silicon-based mineral micro-nano particles act on fibroblasts and vascular endothelial cells inoculated on the stent to promote vascularization and further accelerate skin tissue regeneration. That is, the three-dimensional scaffold formed by using the silicon-based active ink not only has a three-dimensional porous structure, but also has high bioactivity, and can be used for treating skin burn/scald wound surfaces.

In summary, the invention provides a flexible three-dimensional porous scaffold compounded with natural silicon-based mineral micro-nano particles, which realizes the effective recycling of low-cost biological source nonmetallic minerals. The natural diatomite micro-nano particles with special nano porous structures are doped in the hollow porous scaffold, so that the prepared flexible three-dimensional porous scaffold has good air permeability, bioactivity, hydrophilicity and protein adsorption capacity, and has important significance for air exchange, hemostasis, moisture retention, cell migration and differentiation in the skin wound healing process. Meanwhile, the nano holes of the diatomite can be used for carrying medicines or biomolecules, so that the stent has multifunction, and the release of bioactive ions, medicine molecules and the like can be realized at the same time.

The gel contained in the natural inorganic silicon-based material composite three-dimensional scaffold is preferably methacryloylated gelatin. The concentration of the methacryloylated gelatin is preferably 6% on the basis of ensuring printability and moldability.

It should be noted that, at present, research on diatomaceous earth in the field of tissue engineering is mostly focused on the direction of bone regeneration. The invention innovatively selects the low-cost and easily-obtained high-performance natural silicon-based material diatomite to be used for bioactive ink and a composite three-dimensional bracket, and can be used for regenerating skin tissues of severe burn wounds. According to the invention, diatomite is integrated with the 3D printing porous scaffold, the diatomite is used as an inorganic growth factor with stable structure for the first time, and Si ions are released through slow and stable degradation to regulate and control various life activities of dermal fibroblasts and vascular endothelial cells, so that the aim of improving the biological activity of the composite scaffold is fulfilled. The Si ions can promote the expression of the VEGF, an intracellular angiogenesis related factor, and have positive effects on angiogenesis and collagen deposition in the wound healing process.

The disclosure also provides a preparation method of the natural inorganic silicon-based material composite three-dimensional scaffold. Embedding natural silicon-based mineral micro-nano particles into a gel matrix to obtain the silicon-based bioactive ink. The silicon-based bioactive ink may be refrigerated at 4 ℃ until pregelatinized. And preparing the natural inorganic silicon-based material composite three-dimensional bracket by an extrusion type 3D printing technology, and exposing the printed bracket to blue light for crosslinking and curing. As an example, prior to preparing the silicon-based bioactive ink, a dry screening method screens the natural silicon-based mineral micro-nano particles and/or synthetic gel matrix. The method further comprises the step of seeding the scaffold with fibroblasts and vascular endothelial cells, respectively.

The invention prepares the natural diatomite composite three-dimensional porous hydrogel scaffold for the first time. By an extrusion type 3D printing method, diatomite composite biomaterial ink is deposited layer by layer, blue light is crosslinked after printing is finished, and dermal fibroblasts and vascular endothelial cells are respectively inoculated on the stent.

The following shows a specific preparation method of the natural diatomite composite three-dimensional porous hydrogel scaffold in an embodiment of the present invention:

preparation of diatomite composite ink: the natural diatomaceous earth was sieved using a 500 mesh screen using a dry sieving method. Sterilizing by irradiating diatomite particles subjected to particle size screening for more than 1 hour under ultraviolet light. Then adding sterile phosphate buffer solution, and fully dispersing diatomite particles in the buffer solution by ultrasonic to obtain diatomite dispersion liquid. A certain amount of phenyl-2, 4, 6-trimethyl-benzoyl lithium phosphinate (LAP) photoinitiator is weighed and dissolved in phosphate buffer solution at normal temperature. A certain amount of methacryloylated gelatin was weighed and dissolved in a photoinitiator solution at 65 ℃ protected from light. After it was sufficiently dissolved, the solution was filter sterilized using a 0.22 μm filter. Fully mixing the diatomite dispersion liquid and the methacryloylated gelatin solution in a ratio of 1:1, filling the mixture into a printing feed cylinder, and cooling the mixture in a refrigerator at 4 ℃ for about 30 minutes to form pre-gel, thus obtaining the diatomite composite biomaterial ink. The concentration of the methacryloylated gelatin in the ink is 6-8%, preferably 6%.

3D printing process of diatomite composite three-dimensional bracket: and starting the printer and setting a printing program. The whole preparation process is carried out under aseptic conditions by using a cooling printing channel of an extrusion type 3D printer. And loading the cooled diatomite composite biological material ink cartridge into a cooling printing channel, and printing layer by layer under the pushing of air pressure. The temperature of the cooling printing channel is set to be 10 ℃, and each layer rotates by 90 degrees, so that the three-dimensional bracket with the square porous structure is finally formed. After printing, the bracket is exposed to blue light for more than 45 seconds for full crosslinking and curing. The cured scaffold was transferred to a 48-well plate.

Preparation of cells and inoculation: human dermal fibroblasts and human vascular endothelial cells were prepared, and dermal fibroblasts were dispersed in Dulbecco's modified Eagle medium DMEM, and vascular endothelial cells were dispersed in endothelial cell medium ECM. The two cell suspensions are respectively added into different pore plates where the composite bracket is arranged, and the cell density of each pore ranges from 10000 to 30000 cells/pore, preferably 20000 cells/pore. After cell adhesion, the scaffolds were transferred to a new 48-well plate, 1mL DMEM or ECM medium was added to each well, and the well plate was placed in 37℃and 5% CO ₂ Is cultured in a constant temperature incubator every two daysAnd (5) liquid exchange.

The present invention will be further illustrated by the following examples. It is also to be understood that the following examples are given solely for the purpose of illustration and are not to be construed as limitations upon the scope of the invention, since numerous insubstantial modifications and variations will now occur to those skilled in the art in light of the foregoing disclosure. The specific process parameters and the like described below are also merely examples of suitable ranges, i.e., one skilled in the art can make a suitable selection from the description herein and are not intended to be limited to the specific values described below.

Example 1

Screening of natural diatomite: the natural diatomite with smaller particle size is separated and screened by using a 500-mesh screen by using a dry screening method. FIG. 1 shows the result of scanning electron micrographs and X-ray diffraction analysis of diatomite microparticles. The diatomite has a highly ordered nanoscale porous structure, and the diatomite comprises amorphous SiO with higher purity ₂ And (3) phase (C).

Example 2

Biocompatibility of diatomaceous earth

And (3) placing the sieved diatomite under ultraviolet light for irradiation for more than 1 hour for sterilization. The mixture was added to DMEM or ECM medium, respectively, to prepare a stock solution of diatomite dispersion medium having a concentration of 500. Mu.g/mL. By dilution at different multiples, DE-dispersed DMEM/ECM medium was obtained at concentrations of 500. Mu.g/mL, 250. Mu.g/m, 100. Mu.g/mL, 50. Mu.g/mL, 25. Mu.g/mL, 10. Mu.g/mL, respectively.

Human dermal fibroblasts were inoculated into 96-well plates at a density of 500 per well, and cultured by adding 100. Mu.L of diatomaceous earth-dispersed DMEM medium at different concentrations, respectively; human vascular endothelial cells were seeded at a density of 1000 per well in 96-well plates and cultured by adding 100 μl of different concentrations of celite-dispersed ECM medium, respectively. Cell proliferation rates were measured for 1, 3 and 5 days of culture.

FIG. 2 shows proliferation of fibroblasts and vascular endothelial cells within 5 days of culture in a celite dispersion medium. The diatomite has good biocompatibility at lower concentration.

Example 3

Preparation of diatomite composite ink

After the diatomite is screened and separated by a dry screening method, 0.012g, 0.024g, 0.048g and 0.072g are respectively weighed out, and the diatomite is sterilized under ultraviolet light for more than 1 hour. 2mL of phosphate buffer solution was added to each, and the mixture was well dispersed by sonication for 1 hour after sealing. 0.05g of photoinitiator and 1.2g of methacryloylated gelatin were weighed, the photoinitiator was first dissolved in 10mL of phosphate buffer solution at normal temperature, then the methacryloylated gelatin was added, protected from light, and dissolved in a 65℃water bath. After it was completely dissolved, the solution was filter sterilized using a 0.22 μm filter membrane. 2mL of the diatomaceous earth dispersion liquid and 2mL of the methacryloylated gelatin solution were taken and mixed thoroughly, and then cooled at 4℃for about half an hour, whereby diatomaceous earth composite inks (0, 5%, 10%, 20%, 30%) of different concentrations were obtained.

Fig. 3 shows the results of rheological tests of the diatomite composite ink with different diatomite contents. The ink has temperature sensitivity, can be molded under the low-temperature condition, has the shear thinning characteristic, and is suitable for extrusion type 3D printing.

Example 4

Preparation of 3D printing diatomite composite hydrogel three-dimensional scaffold

Step one: preparation of diatomite composite ink

Sieving diatomite by a dry sieving method, respectively weighing 0.012g, 0.024g, 0.048g and 0.072g, and sterilizing under ultraviolet light for more than 1 hour; 2mL of phosphate buffer solution was added to each, and after sealing, the mixture was sonicated for 1 hour to allow sufficient dispersion to give a kieselguhr dispersion. Weighing 0.05g of photoinitiator and 1.2g of methacryloylated gelatin, dissolving the photoinitiator in 10mL of phosphate buffer solution at normal temperature, adding the methacryloylated gelatin, and dissolving in a water bath at 65 ℃ in a dark place; after it was completely dissolved, the solution was sterilized by filtration using a 0.22 μm filter membrane to obtain a methacryloylated gelatin solution. 2mL of the diatomaceous earth dispersion liquid and 2mL of the methacryloylated gelatin solution were taken and mixed thoroughly, and then cooled at 4℃for about half an hour, whereby diatomaceous earth composite inks (0, 5%, 10%, 20%, 30%) having different diatomaceous earth contents were obtained.

Step two: 3D printing process of composite bracket

The printer is started and a printing program is set. The printing support is a square three-dimensional support with a porous structure, the included angle between layers is 90 degrees, and the thickness is about 0.7mm. And loading the cooled feed cylinder filled with the composite ink into a cooling channel of a printer, wherein the temperature of the cooling channel is set to be 10 ℃. The setting range of the extrusion printing air pressure is 40-60kPa, and the type of the extrusion needle head is 27G needle head with the inner diameter of about 250 mu m. After calibrating the needle position, the printing program is started. After printing, exposing the bracket to blue light for more than 45 seconds to fully crosslink and solidify the bracket. And printing ink with different diatomite contents respectively to obtain five diatomite composite hydrogel three-dimensional porous scaffolds Gel, 5DE-Gel, 10DE-Gel, 20DE-Gel and 30DE-Gel.

FIG. 4 shows five diatomite composite three-dimensional porous scaffolds of Gel, 5DE-Gel, 10DE-Gel, 20DE-Gel, 30DE-Gel (left to right). The higher the diatomaceous earth content, the lower the transparency of the scaffold. The microscopic porous structure of the hydrogel can be observed through a scanning electron microscope and an element distribution photo of the section of the bracket, and the inner wall of the internal pore of the bracket is gradually roughened from smooth along with the increase of the concentration of diatomite. The diatomite is uniformly distributed in the bracket, and no obvious agglomeration exists.

Example 5

3D printing diatomite composite hydrogel three-dimensional bracket inoculated with fibroblasts

Step one: preparation of diatomite composite ink

Step two: 3D printing process of composite bracket

The printer is started and a printing program is set. The printing support is a square three-dimensional support with a porous structure, the included angle between layers is 90 degrees, and the thickness is about 0.7mm. And loading the cooled feed cylinder filled with the composite ink into a cooling channel of a printer, wherein the temperature of the cooling channel is set to be 10 ℃. The setting range of the extrusion printing air pressure is 40-60kPa, and the type of the extrusion needle head is 27G needle head with the inner diameter of about 250 mu m. After calibrating the needle position, the printing program is started. After printing, exposing the bracket to blue light for more than 45 seconds to fully crosslink and solidify the bracket. And printing ink with different diatomite contents respectively to obtain five diatomite composite hydrogel three-dimensional porous scaffolds Gel, 5DE-Gel, 10DE-Gel, 20DE-Gel and 30DE-Gel. The rack was transferred into a 48-well plate.

Step three: inoculation of human dermal fibroblasts

The cultured human dermal fibroblasts were digested with trypsin and uniformly dispersed in DMEM medium to prepare a cell suspension. Cell counting is performed on the cell suspension to determine the concentration of cells in the suspension. Fibroblast cells were seeded on the scaffold surface in 48 well plates at a cell concentration of 10000 cells/well, after cell adhesion, the scaffold was transferred to a new 48 well plate, 1mL DMEM medium was added to each well, and placed in an incubator for culture. Proliferation of fibroblasts was characterized by CCK-8 assay kit for 1, 3, and 5 days of culture.

FIG. 5 is a fluorescence micrograph of the distribution of fibroblasts on the surface of a scaffold after 1 day and 5 days of culture, and proliferation of fibroblasts at 1, 3, 5 days. The fibroblasts on the surface of the scaffold proliferate and migrate rapidly during the culture process, eventually covering the entire scaffold.

Example 6

Three-dimensional stent of diatomite composite hydrogel for 3D printing inoculated with vascular endothelial cells

Step one: preparation of diatomite composite ink

Step two: 3D printing process of composite bracket

Step three: inoculation of human vascular endothelial cells

The cultured human vascular endothelial cells are digested by trypsin and uniformly dispersed in an ECM culture medium to prepare a cell suspension. Cell counting is performed on the cell suspension to determine the concentration of cells in the suspension. Vascular endothelial cells were seeded onto the surface of the scaffolds in the 48-well plate at a cell concentration of 20000 cells/well, and after cell adhesion, the scaffolds were transferred to a new 48-well plate, 1mL of ECM medium was added to each well, and placed in an incubator for culturing. Proliferation of vascular endothelial cells was characterized by CCK-8 assay kit at 1, 3, 5 days of culture.

FIG. 6 is a fluorescence micrograph of vascular endothelial cells distributed on the surface of a scaffold after 1 day of culture and 5 days of culture. Comparison of Gel scaffolds without diatomaceous earth can be found that the addition of diatomaceous earth improved the adhesion and spreading of vascular endothelial cells on the scaffold surface. The cell adhesion rate on the 5DE-Gel and 10DE-Gel scaffolds is significantly higher than that of the other three groups, and from the statistical result of cell spreading area, diatomite doped in the scaffolds provides more attachment sites for cells, thus greatly improving the spreading degree of cells. Meanwhile, the 5DE-Gel can obviously promote proliferation of vascular endothelial cells on the stent. Since the diatomite content in the 30DE-Gel scaffold is too high, which is unfavorable for maintaining the cell activity, 1-20% is determined to be a proper concentration range of the diatomite in the composite scaffold.

Example 7

Vasogenic activity of 3D printing diatomite composite hydrogel three-dimensional scaffold

Step one: preparation of diatomite composite ink

Sieving diatomite by a dry sieving method, respectively weighing 0.012g, 0.024g, 0.048g and 0.072g, and sterilizing under ultraviolet light for more than 1 hour; 2mL of phosphate buffer solution was added to each, and after sealing, the mixture was sonicated for 1 hour to allow sufficient dispersion to give a kieselguhr dispersion. Weighing 0.05g of photoinitiator and 1.2g of methacryloylated gelatin, dissolving the photoinitiator in 10mL of phosphate buffer solution at normal temperature, adding the methacryloylated gelatin, and dissolving in a water bath at 65 ℃ in a dark place; after it was completely dissolved, the solution was sterilized by filtration using a 0.22 μm filter membrane to obtain a methacryloylated gelatin solution. 2mL of the diatomaceous earth dispersion liquid and 2mL of the methacryloylated gelatin solution were taken and mixed thoroughly, and then cooled at 4℃for about half an hour, whereby diatomaceous earth composite inks (0, 5%, 10%, 20%) having different diatomaceous earth contents were obtained.

Step two: 3D printing process of composite bracket

The printer is started and a printing program is set. The printing support is a square three-dimensional support with a porous structure, the included angle between layers is 90 degrees, and the thickness is about 0.7mm. And loading the cooled feed cylinder filled with the composite ink into a cooling channel of a printer, wherein the temperature of the cooling channel is set to be 10 ℃. The setting range of the extrusion printing air pressure is 40-60kPa, and the type of the extrusion needle head is 27G needle head with the inner diameter of about 250 mu m. After calibrating the needle position, the printing program is started. After printing, exposing the bracket to blue light for more than 45 seconds to fully crosslink and solidify the bracket. And printing the ink with different diatomite contents respectively to obtain four diatomite composite hydrogel three-dimensional porous scaffolds Gel, 5DE-Gel, 10DE-Gel and 20DE-Gel. The rack was transferred into a 48-well plate.

Step three: inoculation of human vascular endothelial cells

The cultured human vascular endothelial cells are digested by trypsin and uniformly dispersed in an ECM culture medium to prepare a cell suspension. Cell counting is performed on the cell suspension to determine the concentration of cells in the suspension. Vascular endothelial cells were seeded onto the surface of the scaffolds in the 48-well plate at a cell concentration of 20000 cells/well, and after cell adhesion, the scaffolds were transferred to a new 48-well plate, 1mL of ECM medium was added to each well, and placed in an incubator for culturing.

Step four: detection of angiogenesis-related gene expression and ion release

After vascular endothelial cells are inoculated, the stent culture medium is collected after culturing for 1, 2, 3 and 5 days, and the concentration of Si ions in the culture medium is measured by inductively coupled plasma emission spectrometry ICP-AES after filtering. On the fifth day of culture, the RT-PCR method was used to characterize the expression of the angiogenic-related genes in the cells within the scaffold. The specific operation is as follows: vascular endothelial cells on the scaffolds were digested with trypsin and centrifuged, and after the supernatant was aspirated, 1mL Trizol reagent was added to the lower pellet to extract RNA. After purification, RNA was reverse transcribed into cDNA by PrimeScript 1st Strand kit, and RT-qPCR was performed using SYBR Green kit. Finally according to 2 ^-ΔΔCt The method processes the data to obtain the relative expression level of the genes related to the blood vessels.

FIG. 7 shows Si ion release curves of five scaffolds during culture and expression levels of the angiogenized genes on four scaffolds of Gel, 5DE-Gel, 10DE-Gel, 20DE-Gel. In the culture process, diatomite microparticles inside the stent slowly degrade and gradually release bioactive Si ions, the Si ions in a certain concentration range can stimulate vascular endothelial cells, and the expression of genes related to angiogenesis including VEGF, HIF-1 alpha, VE-cad, KDR and the like is obviously promoted. Taken together, the 5DE-Gel scaffold had the highest contributing vascular activity. Thus, a composite three-dimensional scaffold with 5% diatomaceous earth content is most preferred.

The results show that the 3D printing natural diatomite composite three-dimensional porous scaffold has good biological performance of promoting skin cell adhesion, migration, proliferation and vascularization, and has potential application value in promoting skin tissue regeneration and accelerating wound repair.

Example 8

3D printing diatomite composite hydrogel three-dimensional stent for treating severe scalds

Step one: preparation of diatomite composite ink

Sieving diatomite by a dry sieving method, separating, weighing 0.012g, and sterilizing under ultraviolet light for more than 1 hour; 2mL of phosphate buffer solution was added, and after sealing, the mixture was subjected to ultrasonic treatment for 1 hour to sufficiently disperse the mixture, thereby obtaining a diatomaceous earth dispersion. Weighing 0.05g of photoinitiator and 1.2g of methacryloylated gelatin, dissolving the photoinitiator in 10mL of phosphate buffer solution at normal temperature, adding the methacryloylated gelatin, and dissolving in a water bath at 65 ℃ in a dark place; after it was completely dissolved, the solution was sterilized by filtration using a 0.22 μm filter membrane to obtain a methacryloylated gelatin solution. 2mL of the diatomaceous earth dispersion and 2mL of the methacryloylated gelatin solution were taken and mixed thoroughly, and cooled at 4℃for about half an hour, to obtain a diatomaceous earth composite ink having a concentration of 5%.

2mL of phosphate buffer solution and 2mL of methacryloylated gelatin solution were mixed thoroughly, and cooled at 4℃for about half an hour, to obtain a diatomite-free hydrogel ink.

Step two: 3D printing process of composite bracket

The printer is started and a printing program is set. The printing support is a round three-dimensional support with a porous structure, the included angle between layers is 90 degrees, the diameter is 1cm, and the thickness is about 0.7mm. The cooled cartridge containing the pregelatinized ink was loaded into the printer cooling tunnel, which was set to 10 ℃. The setting range of the extrusion printing air pressure is 40-60kPa, and the type of the extrusion needle head is 27G needle head with the inner diameter of about 250 mu m. After calibrating the needle position, the printing program is started. After printing, exposing the bracket to blue light for more than 45 seconds to fully crosslink and solidify the bracket. And printing ink with different diatomite contents respectively to obtain two three-dimensional porous supports Gel and 5DE-Gel.

Step three: establishment and treatment of severe scald model

A second-level scalding model was established on the back of male BALB/c mice (6-8 weeks, SPF clean grade). After anesthetizing the mice, the back hair of the mice was removed, the back skin was exposed and sterilized. Immersing a metal rod with the section diameter of 1cm in boiling water at the temperature of 100 ℃, taking out, and pressing the metal rod on the back skin of a mouse for 5 seconds to form a round secondary scald wound surface. Mice were divided into three groups: blank, gel scaffold, and 5DE-Gel scaffold. After the prepared stent is applied to the scalded part, the stent is fixed by using a medical adhesive tape, a blank group is not treated, and the medical adhesive tape is directly adhered to the wound surface. Surgery when the diary was day 0, the wound was recorded and the scaffold was replaced on days 0, 2, 5, 8, 11, 14. The relative wound area at each time point was counted. Skin tissue at the wound site was harvested after 14 days and analyzed histologically.

Fig. 8 shows the wound healing within 14 days of each group of scalded wounds. Wounds treated with the diatomite composite three-dimensional scaffold exhibited the fastest skin repair rate, and had substantially completely healed by 14 days. According to the tissue staining results, eschar was also present in the blank and Gel groups, re-epithelialization was incomplete, whereas the 5DE-Gel group neonatal skin tissue had formed intact dermal and epidermal tissue. Meanwhile, the 5DE-Gel stent can obviously promote angiogenesis and collagen deposition in new skin tissues, thereby improving the healing efficiency of scalded wound surfaces.

The results show that the 3D printed natural diatomite composite three-dimensional scaffold can play a positive role in vascularization, collagen synthesis and skin tissue regeneration in the wound healing process, and has great application potential in the treatment of scalded wound.

Claims

1. The silicon-based bioactive ink is characterized by comprising a gel matrix and silicon-based mineral micro-nano particles embedded in the gel matrix, wherein the mass of the silicon-based mineral micro-nano particles is 1-20% of that of the gel matrix, the silicon-based mineral micro-nano particles are diatomite micro-nano particles, and the particle size of the silicon-based mineral micro-nano particles is 5-15 mu m; the preparation method of the silicon-based bioactive ink comprises the following steps: dispersing the silicon-based mineral micro-nano particles in a solvent to form silicon-based mineral micro-nano particle dispersion liquid with the mass fraction of the silicon-based mineral micro-nano particles of 0.1-3.0%; dispersing a gel matrix or the gel matrix and a photoinitiator in a solvent to form a gel solution with the mass fraction of the gel matrix of 12-16%; uniformly mixing the dispersion liquid and the gel solution of the hard silicon-based mineral micro-nano particles to obtain the silicon-based bioactive ink.

2. The silicon-based bioactive ink of claim 1 wherein the gel matrix comprises one or more of hyaluronic acid gel, methacryloylated gelatin, sodium alginate gel.

3. The silicon-based bioactive ink of claim 1, wherein the mass of the hard silicon-based mineral micro-nano particles is 5-20% of the gel matrix.

4. The silicon-based bioactive ink of claim 1 wherein the volume ratio of the hard silicon-based mineral micro-nanoparticle dispersion to the gel solution is 1:1.

5. The silicon-based bioactive ink of claim 1 wherein the solvent is independently selected from at least one of deionized water, ultrapure water, phosphate buffer solution.

6. A flexible three-dimensional porous scaffold made of natural inorganic silicon-based materials, which is characterized by being formed by cross-linking and solidifying the silicon-based bioactive ink according to any one of claims 1-5 after 3D printing; the natural inorganic silicon-based material flexible three-dimensional porous scaffold has a regular porous structure, and comprises a basic framework formed by solidifying the gel matrix and the hard silicon-based mineral micro-nano particles which are embedded into the framework and used as active factors for regulating cell behaviors, wherein the mass of the hard silicon-based mineral micro-nano particles is 1-20% of that of the gel matrix.

7. Use of a flexible three-dimensional porous scaffold of a natural inorganic silicon-based material according to any one of claims 1 to 5 or of a natural inorganic silicon-based material according to claim 6 for the preparation of bioengineering materials.

8. The use according to claim 7, characterized in that it is used in the preparation of a medicament for the treatment of burns and scalds.