CN114870071A

CN114870071A - Silicon-based bioactive ink, natural inorganic silicon-based material flexible three-dimensional porous scaffold and application

Info

Publication number: CN114870071A
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: 2022-08-09
Anticipated expiration: 2042-04-29
Also published as: CN114870071B

Abstract

The invention discloses silicon-based bioactive ink, a natural inorganic silicon-based material flexible three-dimensional porous support and application. 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 that of the gel matrix, and is preferably 1-20%. The flexible three-dimensional porous scaffold made of the natural inorganic silicon-based material has a regular porous structure, and the hard silicon-based mineral micro-nano particles in the flexible three-dimensional porous scaffold can be slowly degraded and release bioactive ions to promote angiogenesis and further accelerate skin repair, so that the flexible three-dimensional porous scaffold has an important significance for treating severe burns and scalds.

Description

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

Technical Field

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

Background

The skin is the first barrier for protecting the internal environment of the human body, and plays an important role in maintaining the body temperature, preventing water loss, regulating the metabolic process, resisting external invasion and the like. High temperature, chemical corrosion, electric shock, etc. all cause severe burns to the skin. Severe burns can result in the destruction of the vascular network and dermal matrix within the skin tissue, in which case the difficulty of skin healing increases greatly and may worsen to chronic wounds. Thus, there is a need to develop multifunctional wound dressings that can support gas exchange, retain wound moisture, and promote angiogenesis and collagen deposition to accelerate skin tissue regeneration in burn wounds.

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 ingrowth 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 a silicon-based bioactive ink, a natural inorganic silicon-based material flexible three-dimensional porous scaffold and application, wherein the natural inorganic silicon-based material flexible three-dimensional porous scaffold has a regular porous structure, and hard (natural) silicon-based mineral micro-nano particles in the natural inorganic silicon-based material can be slowly degraded and release bioactive ions to promote angiogenesis so as to accelerate skin repair, so that the silicon-based bioactive ink has important significance for treating severe burn and scald wounds.

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 that of the gel matrix, and is preferably 1-20%. The quality of the hard silicon-based mineral micro-nano particles is within the range, bioactive ions generated by stable degradation of the hard silicon-based mineral micro-nano particles in the silicon-based bioactive ink can have positive influence on the life activities of skin cells, a bioactive ion microenvironment with proper concentration is established, and cell migration, proliferation and 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, and preferably 5-15 μm.

Preferably, the gel matrix comprises one or more of hyaluronic acid gel, methacrylated gelatin and sodium alginate gel; preferably, the gel matrix is methacrylated 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 any one of the silicon-based bioactive inks described above. Dispersing the hard silicon-based mineral micro-nano particles in a solvent to form a hard silicon-based mineral micro-nano particle dispersion liquid with the mass fraction of the hard silicon-based mineral micro-nano particles being 0.1-3.0%; dispersing the gel matrix or the gel matrix and a photoinitiator in a solvent to form a gel solution with the gel matrix mass fraction 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 the dispersion to the 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 invention provides a natural inorganic silicon-based material flexible three-dimensional porous scaffold (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 base frame formed by solidifying a gel matrix and hard silicon-based mineral micro-nano particles which are embedded in the base frame and used as active factors to regulate cell behaviors, wherein 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%. Preferably, the hard silicon-based mineral micro-nano particles are diatomite micro-nano particles.

Preferably, the flexible three-dimensional porous scaffold made of the natural inorganic silicon-based material is formed by cross-linking and curing silicon-based bioactive ink after 3D printing, 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 an application of the silicon-based bioactive ink or the natural inorganic silicon-based material flexible three-dimensional porous scaffold in bioengineering, especially in treatment of skin scald and burn wound.

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

Drawings

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

FIG. 2 is a representation of the cytocompatibility of diatomaceous earth microparticles, including (a) fibroblasts and (b) vascular endothelial cell proliferation, within 5 days of culture in diatomaceous earth dispersion medium. The bar chart of FIG. 2 is, from left to right, CTR, 10. mu.g/mL, 25. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, 250. mu.g/mL, 500. mu.g/mL.

Fig. 3 is a rheological characterization of the diatomite composite inks with different diatomite contents, which shows that (a) the methacrylated gelatin ink has temperature sensitivity, (b) the diatomite composite ink has temperature sensitivity, and (c) both the methacrylated gelatin ink and the diatomite composite ink have shear thinning performance. (c) The five inks in (1) are very similar in shear thinning behavior so that overlap is inevitable, which does not affect the disclosure of the invention.

FIG. 4 is a representation of 3D printed diatomaceous earth composite three-dimensional scaffolds (Gel, 5DE-Gel, 10DE-Gel, 20DE-Gel, 30DE-Gel), including (a) appearance photographs, (b) internal scanning electron microscope photographs, and (c) Si and O element distribution photographs of the scaffolds. (b) All scales of (2) are 100 μm.

FIG. 5 shows fluorescence micrographs of (a, b) day 1 and day 5 cell distributions of a composite three-dimensional scaffold of diatomaceous earth seeded with fibroblasts and (c) proliferation of fibroblasts on the scaffold within 5 days. (a) The scales of (a) and (b) are each 500. mu.m. (c) The bar graphs are Gel, 5DE-Gel, 10DE-Gel, 20DE-Gel and 30DE-Gel from left to right in sequence.

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

FIG. 7 is a graph showing the (a) angiogenesis-related gene expression and (b) Si ion release profile in a diatomite composite three-dimensional scaffold seeded with vascular endothelial cells. (a) The bar graphs are Gel, 5DE-Gel, 10DE-Gel and 20DE-Gel from left to right in sequence. (b) The lines of (A) are sequentially referred to as Gel, 5DE-Gel, 10DE-Gel, 20DE-Gel and 30DE-Gel from bottom to top.

FIG. 8 is a study of a 5DE-Gel diatomite composite three-dimensional scaffold for skin scald wound treatment, including (a) wound photographs and (b) statistics of relative wound areas within 14 days; histological staining analysis included (c) Masson trichrome staining (collagen fiber: blue) and (d) statistics of collagen fiber content, (e) CD31 immunofluorescence staining (blood vessels: green, nuclei: blue) and (f) statistics of blood vessel numbers. (b) The bar graphs are Blank, Gel and 5DE-Gel from left to right. (a) Scale of (b) is 1cm, scale of (c) is 500 μm and scale of (e) is 200 μm.

Detailed Description

The present invention is further illustrated by the following examples, which are to be understood as merely illustrative of, and not restrictive on, the present invention. Unless otherwise specified, each percentage means a mass percentage.

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 biological performance improvement effect on ink and a bracket is not obvious, the concentration of released bioactive ions is not in an effective range, and the regulation and control on cell behaviors are difficult to realize. If the mass ratio of the hard silicon-based mineral micro-nano particles is higher than 20%, crosslinking of a gel matrix polymer network is affected, the structural stability and the mechanical property of the scaffold are poor, and meanwhile, high-concentration ions released by the excessively high silicon-based mineral micro-nano particles inhibit cell activity and reduce the cell survival rate.

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 ions comprise one or more of Ca, Si, Mg and Zn ions. For example, the bioactive ion is a Si ion.

The hard silicon-based mineral micro-nano particles can be porous natural non-metallic minerals. In some technical schemes, the natural silicon-based mineral micro-nano particles are natural diatomite micro-nano particles. The diatomite is composed ofSkeleton formed by deposition of diatom of unicellular aquatic plant, its main component is SiO ₂ ·nH ₂ And 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 silicon dioxide synthesized by a chemical method, the diatomite does not have the 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 cell life activities.

The invention provides the bioactive ink containing the 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 printable performance and forming performance and can be cured and crosslinked under the irradiation of blue light. While there are many natural silica-based minerals, the choice of diatomaceous earth in the present invention is due to its performance advantages not available with other silica-based minerals. The diatomite is a non-metallic mineral of biological origin, has pH near neutrality, is non-toxic, is insoluble in most acids, and has good biocompatibility and stability. In addition, compared with other silicon-based minerals such as silicate minerals, highly regular and dense nano-pore structures are distributed on the diatomite micro-nano particles, so that strong adsorption performance is given to the diatomite. The natural diatomite composite ink and the bracket have high water content and water retention due to excellent water absorption performance, which are required by the skin burn wound dressing, can keep a relatively humid environment for the wound and are beneficial to wound healing. Meanwhile, a large number of hydroxyl groups on the surface of the natural diatomite can also enhance the protein adsorption capacity of the ink and the bracket, and is also beneficial to promoting the skin repair process. This is reflected in the ability of diatomaceous earth to 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 less than 20 mu m. The purpose of controlling the particle size of the hard silicon-based mineral micro-nano particles within the range is to ensure that the addition of the hard silicon-based mineral micro-nano particles does not influence the printable performance of the ink and the gel matrix forming performance, for example, agglomeration and blockage do not occur in the extrusion printing process. 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 less than 20 microns. 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 methacrylated gelatin (GelMA). The methacrylated gelatin has good biocompatibility, and the degradation product has no cytotoxicity.

In some technical schemes, the mass of the hard diatomite micro-nano particles is 5-20% of that of the methacryloylated gelatin. Thus, the printability and photocrosslinking performance of the ink can be further improved, and the situation that the released ions are high in concentration and are not beneficial to cell survival is avoided.

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

In some technical schemes, the diatomite powder is soaked in a solvent and is prepared into the diatomite dispersion liquid through ultrasonic dispersion. Methacrylated gelatin (GelMA) and a photoinitiator were dissolved in a solvent at 65 ℃ to obtain a GelMA hydrogel solution. Fully mixing the diatomite dispersion liquid with the GelMA hydrogel solution, and cooling at 4 ℃ to form the printable silicon-based bioactive ink. Before the diatomite is used, the diatomite from the diatomite deposition outer framework is subjected to particle size separation through a dry screening method, specifically, a 500-mesh screen is used for screening diatomite powder in a dry state, and screened particles are sterilized under ultraviolet light for more than 1 hour and used for preparing bioactive ink.

The present disclosure also provides a natural inorganic silica-based material composite three-dimensional scaffold (natural inorganic silica-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 the flexible three-dimensional porous bracket for the first time. The composite three-dimensional scaffold comprises a base framework formed by solidifying a gel matrix and hard silicon-based mineral micro-nano particles which are embedded in the framework and used as active factors to regulate cell behaviors. The diatomite has light weight, low density, high hydrophilicity and high adsorbability, mainly comes from a unique regular nano porous structure naturally formed by the diatomite and silicon hydroxyl distributed on the surface of the diatomite, can enhance the mechanical property, the hydrophilicity and the surface roughness of the stent, endows the stent with good water absorbability and protein adsorbability, and does not influence the pH value and the biocompatibility of the stent. 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 are released through degradation to stimulate the cells to spread, migrate, proliferate and differentiate. The scaffold can be used as a scald/burn wound dressing, accelerates skin repair by promoting angiogenesis, and has great application potential in the treatment of severe burns and scalds of skin wounds.

By way of 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 glue, and has a regular porous structure. During the culture process, the natural silicon-based mineral micro-nano particles in the stent are slowly degraded to release Si ions with proper concentration, which act on fibroblasts and vascular endothelial cells inoculated on the stent, so that vascularization is promoted, and the regeneration of skin tissues is accelerated. Namely, the three-dimensional scaffold formed by using the silicon-based active ink not only has a three-dimensional porous structure, but also has high biological activity, and can be used for treating skin burn/scald wounds.

In conclusion, the invention provides the flexible three-dimensional porous support compounded with the natural silicon-based mineral micro-nano particles, and the effective reutilization of the low-cost biological non-metallic minerals is realized. Because the natural diatomite micro-nano particles with special nano-porous structures are doped in the flexible three-dimensional porous scaffold, the prepared flexible three-dimensional porous scaffold has good air permeability, biological activity, hydrophilicity and protein adsorption capacity, and has important significance on gas exchange, hemostasis, water retention, cell migration and differentiation in the skin wound healing process. Meanwhile, the nano-pores of the diatomite can be used for carrying out drug or biomolecule loading, thus endowing the stent with multifunction and simultaneously realizing the release of bioactive ions, drug molecules and the like.

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

It is worth mentioning that the research on diatomaceous earth in the field of tissue engineering is currently focused on the direction of bone regeneration. The invention creatively selects the natural silicon-based material diatomite with low cost and easy acquisition and high performance 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, the diatomite and the 3D printing porous scaffold are integrated, the diatomite is used as an inorganic 'growth factor' with a stable structure for the first time, Si ions are released through slow and stable degradation to regulate various life activities of dermal fibroblasts and vascular endothelial cells, and the purpose of improving the biological activity of the composite scaffold is achieved. Si ions can promote the expression of an intracellular angiogenesis-related factor VEGF, and have a positive effect 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 bracket. And embedding the natural silicon-based mineral micro-nano particles into a gel matrix to obtain the silicon-based bioactive ink. The silicon-based bioactive ink can be refrigerated at 4 ℃ until pre-gelation occurs. Preparing a 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, before preparing the silicon-based bioactive ink, a dry sieving method screens natural silicon-based mineral micro-nano particles and/or synthetic gel matrix. The method further comprises the step of seeding fibroblasts and vascular endothelial cells on the scaffold respectively.

The invention prepares the natural diatomite composite three-dimensional porous hydrogel scaffold for the first time. The diatomite composite biological material ink is deposited layer by layer through an extrusion type 3D printing method, blue light crosslinking is carried out 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 one embodiment of the present invention:

preparing the diatomite composite ink: the natural diatomaceous earth was sieved using a 500 mesh sieve using a dry sieving method. And irradiating the diatomite particles with the screened particle sizes for more than 1 hour under ultraviolet light for sterilization. And then adding a sterile phosphate buffer solution, and performing ultrasonic treatment to fully disperse the diatomite particles in the buffer solution to obtain the diatomite dispersion solution. Weighing a certain amount of lithium phenyl-2, 4, 6-trimethyl-benzoylphosphinate (LAP) photoinitiator, and dissolving the photoinitiator in a phosphate buffer solution at normal temperature. Weighing a certain amount of methacrylated gelatin, and dissolving the methacrylated gelatin in a photoinitiator solution at 65 ℃ in the dark. After it was sufficiently dissolved, the solution was filter sterilized using a 0.22 μm filter. And (3) fully mixing the diatomite dispersion liquid and the methacrylated gelatin solution in a ratio of 1:1, filling the mixture into a printing cylinder, and cooling the mixture in a refrigerator at 4 ℃ for about 30 minutes to form a pre-gel, thus obtaining the diatomite composite biological material ink. The concentration of the methacrylated gelatin in the ink is 6 to 8%, preferably 6%.

3D printing process of the diatomite composite three-dimensional support: the printer and the settings of the printing program are started. The cooling printing channel of the extrusion type 3D printer is used in the preparation process, and the whole preparation process is carried out under the aseptic condition. And (3) filling the cooled diatomite composite biological material ink cylinder 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 ℃, the rotation angle of each layer is 90 degrees, and finally the three-dimensional support with the square porous structure is formed. And after printing, exposing the bracket under blue light for more than 45 seconds to fully crosslink and solidify. The cured scaffolds were transferred to 48-well plates.

Preparation and inoculation of cells: 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 on which the composite scaffold is arranged, and the cell density of each pore is 10000-30000/pore, preferably 20000/pore. After the cells adhered, the scaffolds were transferred to a new 48-well plate, 1mL DMEM or ECM medium was added to each well, and the plate was placed at 37 ℃ and 5% CO ₂ The culture is carried out in a constant temperature incubator, and the liquid is changed every two days.

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 may be made in light of the above teachings. 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

Screening 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 micrograph and X-ray diffraction analysis of the diatomaceous earth micron particles. The diatomite has a highly regular and ordered nano-scale porous structure and comprises amorphous SiO with higher purity ₂ And (4) phase(s).

Example 2

Biocompatibility of diatomaceous earth

Sterilizing the sieved diatomite by irradiating the diatomite for more than 1 hour under ultraviolet light. The solutions were added to DMEM or ECM medium, respectively, to prepare a 500. mu.g/mL diatomaceous earth dispersion medium stock solution. The 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, and 10. mu.g/mL by dilution with different fold.

Inoculating human dermal fibroblasts into a 96-well plate at the density of 500 cells/well, and respectively adding 100 mu L of diatomite-dispersed DMEM (DMEM) culture medium with different concentrations for culture; human vascular endothelial cells were seeded in a 96-well plate at a density of 1000 cells/well, and 100. mu.L of diatomaceous earth-dispersed ECM medium of different concentrations was added thereto, respectively, for culture. The cell growth rate was measured for 1, 3, and 5 days of culture.

FIG. 2 shows the proliferation of fibroblasts and vascular endothelial cells within 5 days of the culture in the diatomaceous earth dispersion medium. Proves that the diatomite has good biocompatibility at lower concentration.

Example 3

Preparation of diatomite composite ink

Sieving diatomaceous earth by dry sieving method, weighing 0.012g, 0.024g, 0.048g, and 0.072g, and sterilizing under ultraviolet light for more than 1 hr. 2mL of each phosphate buffer solution was added, and the mixture was sealed and then sonicated for 1 hour to disperse the mixture sufficiently. 0.05g of photoinitiator and 1.2g of methacrylated gelatin are weighed, the photoinitiator is dissolved in 10mL of phosphate buffer solution at normal temperature, then the methacrylated gelatin is added, and the mixture is kept out of the sun 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 and 2mL of the methacrylated gelatin solution were mixed thoroughly, and then cooled at 4 ℃ for about half an hour to obtain diatomaceous earth composite inks (0, 5%, 10%, 20%, 30%) having different concentrations.

Figure 3 shows the results of rheological testing of the diatomaceous earth composite inks for different diatomaceous earth contents. The ink has temperature sensitivity, can be formed at low temperature, has the shear thinning characteristic of the five inks, and is suitable for extrusion type 3D printing.

Example 4

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

The method comprises the following steps: preparation of diatomite composite ink

Sieving diatomaceous earth by dry sieving method, weighing 0.012g, 0.024g, 0.048g, and 0.072g, sterilizing under ultraviolet light for more than 1 hr; each 2mL of a phosphate buffer solution was added, and the mixture was sealed and then sufficiently dispersed by sonication for 1 hour to obtain 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, keeping out of the sun, and dissolving in a 65 ℃ water bath kettle; after the solution is completely dissolved, the solution is subjected to filtration sterilization by using a 0.22 mu m filter membrane to obtain the methacrylated gelatin solution. 2mL of the diatomaceous earth dispersion and 2mL of the methacrylated gelatin solution were mixed thoroughly, and then cooled at 4 ℃ for about half an hour to obtain diatomaceous earth composite inks (0, 5%, 10%, 20%, 30%) having different diatomaceous earth contents.

Step two: 3D printing process of composite stent

And (5) starting the printer and setting a printing program. 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 0.7 mm. And (3) loading the cooled material cylinder filled with the composite ink into a printer cooling channel, wherein the temperature of the cooling channel is set to be 10 ℃. The extrusion printing pressure was set in the range of 40-60kPa, and the extrusion needle type was a 27G needle having an inner diameter of about 250 μm. After the position of the needle is calibrated, the printing program is started. And after printing is finished, exposing the bracket in blue light for more than 45 seconds to fully crosslink and solidify the bracket. And respectively printing inks with different diatomite contents to obtain five diatomite composite hydrogel three-dimensional porous scaffolds Gel, 5DE-Gel, 10DE-Gel, 20DE-Gel and 30 DE-Gel.

FIG. 4 shows five kinds of diatomite composite three-dimensional porous scaffolds, i.e., Gel, 5DE-Gel, 10DE-Gel, 20DE-Gel and 30DE-Gel (from 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 picture of the cross section of the bracket, and the inner walls of pores in the bracket gradually become rough from smooth along with the increase of the concentration of the diatomite. The diatomite is uniformly distributed in the bracket without obvious agglomeration.

Example 5

3D of inoculation fibroblast prints compound hydrogel three-dimensional support of diatomaceous earth

Step two: 3D printing process of composite stent

And (5) starting the printer and setting a printing program. 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 0.7 mm. And (3) loading the cooled material cylinder filled with the composite ink into a printer cooling channel, wherein the temperature of the cooling channel is set to be 10 ℃. The extrusion printing pressure was set in the range of 40-60kPa, and the extrusion needle type was a 27G needle having an inner diameter of about 250 μm. After the position of the needle is calibrated, the printing program is started. And after printing is finished, exposing the bracket in blue light for more than 45 seconds to fully crosslink and solidify the bracket. And respectively printing inks with different diatomite contents to obtain five diatomite composite hydrogel three-dimensional porous scaffolds Gel, 5DE-Gel, 10DE-Gel, 20DE-Gel and 30 DE-Gel. The scaffolds were transferred to 48-well plates.

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. The cell suspension was cell counted to determine the cell concentration in the suspension. The fibroblast cells are inoculated on the surface of the scaffold in the 48-well plate at the cell concentration of 10000 cells/well, after the cells are adhered, the scaffold is transferred to a new 48-well plate, 1mL of DMEM medium is added into each well, and the mixed solution is placed into an incubator for culture. The proliferation condition of the fibroblasts is characterized by a CCK-8 detection kit on 1, 3 and 5 days of culture.

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

Example 6

3D printing diatomite composite hydrogel three-dimensional stent for inoculating vascular endothelial cells

Step two: 3D printing process of composite stent

Step three: inoculation of human vascular endothelial cells

The cultured human vascular endothelial cells were digested with trypsin and uniformly dispersed in an ECM medium to prepare a cell suspension. The cell suspension was cell counted to determine the cell concentration in the suspension. The vascular endothelial cells were seeded on the surface of the scaffold in 48-well plates at a cell concentration of 20000 cells/well, and after the cells were adhered, the scaffold was transferred to a new 48-well plate, 1mL of ECM medium was added per well, and placed in an incubator for culture. And (5) characterizing the proliferation condition of the vascular endothelial cells by a CCK-8 detection kit on 1, 3 and 5 days of culture.

FIG. 6 is a fluorescent micrograph of the distribution of vascular endothelial cells on the surface of the stent after 1 day and 5 days of culture. It can be seen by comparison of Gel scaffolds without diatomaceous earth that the addition of diatomaceous earth improves the adhesion and spreading of vascular endothelial cells on the surface of the scaffold. The cell adhesion rate of the 5DE-Gel and 10DE-Gel scaffolds is obviously higher than that of the other three groups, and from the statistical result of cell spreading areas, the diatomite doped in the scaffolds provides more attachment sites for cells, so that the spreading degree of the cells is greatly improved. Meanwhile, the 5DE-Gel can obviously promote the proliferation of vascular endothelial cells on the stent. Since the content of the diatomite in the 30DE-Gel scaffold is too high to be beneficial to maintaining the cell activity, 1-20% is determined as the proper concentration range of the diatomite in the composite scaffold.

Example 7

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

Sieving diatomaceous earth by dry sieving method, weighing 0.012g, 0.024g, 0.048g, and 0.072g, sterilizing under ultraviolet light for more than 1 hr; each 2mL of a phosphate buffer solution was added, and the mixture was sealed and then sufficiently dispersed by sonication for 1 hour to obtain 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, keeping out of the sun, and dissolving in a 65 ℃ water bath kettle; after the solution is completely dissolved, the solution is subjected to filtration sterilization by using a 0.22 mu m filter membrane to obtain the methacrylated gelatin solution. 2mL of the diatomaceous earth dispersion and 2mL of the methacrylated gelatin solution were each mixed thoroughly, and then cooled at 4 ℃ for about half an hour to obtain diatomaceous earth composite inks (0, 5%, 10%, 20%) having different diatomaceous earth contents.

Step two: 3D printing process of composite stent

And (5) starting the printer and setting a printing program. 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 0.7 mm. And (3) loading the cooled material cylinder filled with the composite ink into a printer cooling channel, wherein the temperature of the cooling channel is set to be 10 ℃. The extrusion printing pressure was set in the range of 40-60kPa, and the extrusion needle type was a 27G needle having an inner diameter of about 250 μm. After the position of the needle is calibrated, the printing program is started. And after printing is finished, exposing the bracket in blue light for more than 45 seconds to fully crosslink and solidify the bracket. And respectively printing inks with different diatomite contents to obtain four diatomite composite hydrogel three-dimensional porous scaffolds Gel, 5DE-Gel, 10DE-Gel and 20 DE-Gel. The scaffolds were transferred to 48-well plates.

Step three: inoculation of human vascular endothelial cells

The cultured human vascular endothelial cells were digested with trypsin and uniformly dispersed in an ECM medium to prepare a cell suspension. The cell suspension was cell counted to determine the cell concentration in the suspension. The vascular endothelial cells were seeded on the surface of the scaffold in 48-well plates at a cell concentration of 20000 cells/well, and after the cells were adhered, the scaffold was transferred to a new 48-well plate, 1mL of ECM medium was added per well, and placed in an incubator for culture.

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

After the vascular endothelial cells are inoculated, collecting the scaffold culture medium when the culture is carried out for 1, 2, 3 and 5 days, and measuring the concentration of Si ions in the culture medium by inductively coupled plasma emission spectroscopy (ICP-AES) after filtration. Characterization of scaffolds by RT-PCR method at day five of cultureThe expression of the angiogenesis-related genes in the inner cells. The specific operation is as follows: the vascular endothelial cells on the scaffolds were digested with trypsin and centrifuged, and after the supernatant was aspirated, 1mL of Trizol reagent was added to the lower pellet to extract RNA. After purification, the RNA was reverse transcribed into cDNA using 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 angiogenesis-related genes.

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

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

Example 8

3D printing diatomite composite hydrogel three-dimensional scaffold for treating severe scald

Sieving diatomite by dry sieving method, weighing 0.012g, and sterilizing under ultraviolet light for more than 1 hr; adding 2mL of phosphate buffer solution, sealing, and performing ultrasonic treatment for 1 hour to fully disperse the solution to obtain a diatomite dispersion solution. 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, keeping out of the sun, and dissolving in a 65 ℃ water bath kettle; after the solution is completely dissolved, the solution is subjected to filtration sterilization by using a 0.22 mu m filter membrane to obtain the methacrylated gelatin solution. 2mL of the diatomaceous earth dispersion and 2mL of the methacrylated gelatin solution were each mixed thoroughly, and then cooled at 4 ℃ for about half an hour to obtain a 5% diatomaceous earth composite ink.

2mL of the phosphate buffer solution and 2mL of the methacrylated gelatin solution were thoroughly mixed and cooled at 4 ℃ for about half an hour to obtain a hydrogel ink containing no diatomaceous earth.

Step two: 3D printing process of composite stent

And (5) starting the printer and setting a printing program. The printing support is a circular three-dimensional support with a porous structure, the included angle between layers is 90 degrees, the diameter is 1cm, and the thickness is about 0.7 mm. And (3) loading the cooled charging barrel filled with the pre-gel ink into a printer cooling channel, wherein the temperature of the cooling channel is set to be 10 ℃. The pressure setting range of the extrusion type printing is 40-60kPa, and the type of the extrusion needle is a 27G needle with the inner diameter of about 250 μm. After the position of the needle is calibrated, the printing program is started. And after printing is finished, exposing the bracket in blue light for more than 45 seconds to fully crosslink and solidify the bracket. And respectively printing inks with different diatomite contents to obtain two three-dimensional porous scaffolds Gel and 5 DE-Gel.

Step three: establishment and treatment of severe scald model

A secondary scald model was established on the back of male BALB/c mice (6-8 weeks, SPF clean grade). After anesthetizing the mice, the mice were back-haired, and the back skin was exposed and sterilized. A metal rod with the diameter of 1cm of the cross section is immersed in 100 ℃ boiling water, and the metal rod is taken out and tightly pressed on the back skin of the mouse for 5 seconds to form a circular secondary scald wound. The mice were divided into three groups: blank set, Gel scaffold set, and 5DE-Gel scaffold set. After the prepared bracket is pasted on the scalded part, the bracket is fixed by using a medical adhesive tape, and the medical adhesive tape is directly pasted on the wound surface without processing a blank group. The day of surgery was designated as day 0, and the wound surface was recorded and the stent was replaced on

days

0, 2, 5, 8, 11, and 14. Statistics were made of the relative wound area at each time point. Skin tissue from the wound site was harvested 14 days later and subjected to histological analysis.

Fig. 8 shows the healing of wounds within 14 days of the scalded wounds in each group. Wounds treated via the diatomaceous earth composite three-dimensional scaffold exhibited the fastest rate of skin repair, with essentially complete healing at 14 days. According to the tissue staining results, eschar was still present in the blank group and Gel group, re-epithelialization was not completed, while neogenetic skin tissue of 5DE-Gel group had formed intact dermal and epidermal tissue. Meanwhile, the 5DE-Gel stent can remarkably promote angiogenesis and collagen deposition in the new skin tissue, thereby improving the healing efficiency of the scald wound.

The results show that the natural diatomite composite three-dimensional scaffold printed by 3D 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 scald wounds.

Claims

1. The silicon-based bioactive ink is characterized by comprising 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 that of the gel matrix, and is preferably 1-20%.

2. The silicon-based bioactive ink according to claim 1, wherein the hard silicon-based mineral micro-nano particles are diatomite micro-nano particles.

3. The silicon-based bioactive ink according to claim 1 or 2, wherein the hard silicon-based mineral micro-nano particles have a particle size of less than 20 μm, preferably 5-15 μm.

4. The silicon-based bioactive ink according to any one of claims 1 to 3, wherein the gel matrix comprises one or more of hyaluronic acid gel, methacrylated gelatin, sodium alginate gel; preferably, the gel matrix is methacrylated gelatin.

5. The silicon-based bioactive ink according to any one of claims 1 to 4, wherein the mass of the hard silicon-based mineral micro-nano particles is 5-20% of the gel matrix.

6. The preparation method of the silicon-based bioactive ink according to any one of claims 1 to 5, wherein the hard silicon-based mineral micro-nano particles are dispersed in a solvent to form a hard silicon-based mineral micro-nano particle dispersion liquid with the mass fraction of the hard silicon-based mineral micro-nano particles being 0.1-3.0%; dispersing the gel matrix or the gel matrix and a photoinitiator in a solvent to form a gel solution with the gel matrix mass fraction 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 the dispersion to the gel solution is 1: 1.

7. The method for preparing silicon-based bioactive ink according to claim 6, wherein the solvent is independently selected from at least one of deionized water, ultrapure water and phosphate buffer solution.

8. The flexible three-dimensional porous scaffold is characterized by having a regular porous structure and comprising a base frame formed by solidifying a gel matrix and hard silicon-based mineral micro-nano particles which are embedded in the base frame and used as active factors to regulate and control cell behaviors, wherein 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%.

9. The natural inorganic silicon-based material flexible three-dimensional porous scaffold as claimed in claim 8, wherein the natural inorganic silicon-based material flexible three-dimensional porous scaffold is formed by 3D printing of silicon-based bioactive ink, and then cross-linking and curing, and the silicon-based bioactive ink comprises a gel matrix and hard silicon-based mineral micro-nano particles embedded in the gel matrix.

10. Use of the silicon-based bioactive ink according to any one of claims 1 to 5 or the natural inorganic silicon-based material flexible three-dimensional porous scaffold according to any one of claims 8 to 9 for bioengineering, in particular skin scald and burn wound treatment.