CN112426569B

CN112426569B - Inorganic-organic composite living cell scaffold and preparation method and application thereof

Info

Publication number: CN112426569B
Application number: CN202011208524.1A
Authority: CN
Inventors: 吴成铁; 马景阁
Original assignee: Shanghai Institute of Ceramics of CAS
Current assignee: Zhongke Sifukang Jining Medical Device Technology Co ltd
Priority date: 2020-11-03
Filing date: 2020-11-03
Publication date: 2022-02-08
Anticipated expiration: 2040-11-03
Also published as: CN112426569A

Abstract

The invention discloses an inorganic-organic composite living cell scaffold, a preparation method and application thereof. The living cell scaffold comprises a scaffold integral framework formed by constructing inorganic-organic composite biological ink and a plurality of cells which are distributed in a layered manner in the three-dimensional space of the scaffold integral framework; the upper half part of the living cell scaffold is in a layer-by-layer alternating arrangement mode of (A-C) y, wherein A is biological ink, and C is upper-layer cells of the scaffold; the lower half part is in a layer-by-layer alternate arrangement mode of (A-B) x, wherein A is biological ink, and B is cells on the lower layer of the bracket; x and y represent cycle periods which are alternately arranged and are positive integers. The inorganic-organic composite living cell scaffold can simulate the physiological structure of the dermis of human skin containing blood vessels, promote the regeneration of the blood vessels and the reconstruction of the dermis structure so as to accelerate the regeneration of the skin, and has potential application value in the treatment of the full-thickness injury of the skin and the wound surface which is difficult to heal.

Description

Inorganic-organic composite living cell scaffold and preparation method and application thereof

Technical Field

The invention relates to an inorganic-organic composite living cell scaffold, a preparation method and application thereof, in particular to an inorganic-organic composite biological ink-based living cell scaffold with multi-cell layered distribution, a preparation method and application thereof, and belongs to the technical field of biology.

Background

Burns, lacerations, diabetic ulcers and the like in daily life can cause deep skin injuries which are difficult to heal, and the injuries are often accompanied with serious damage to skin blood vessels, so that the human body is difficult to heal. The current clinical best treatment means is autograft or allograft, but the autograft often has the defects of insufficient donor source and secondary wound generation; allograft has immunological rejection. Based on the above, the preparation of a living skin tissue substitute that can be used for transplantation using an artificial method has been urgently required.

In recent years, a newly-emerging biological 3D printing technology can simultaneously print materials and cells, accurately control the distribution of the cells in a three-dimensional space, and realize the artificial preparation of a three-dimensional tissue construct and the application of the three-dimensional tissue construct in organ reconstruction. Therefore, the biological 3D printing technology has the application potential of directly constructing living tissues imitating the skin and dermis in vitro.

Disclosure of Invention

Aiming at the problems, the invention provides an inorganic-organic composite living cell scaffold and a preparation method and application thereof. The inorganic-organic composite living cell scaffold can simulate the physiological structure of the dermis of human skin containing blood vessels, promote the regeneration of the blood vessels and the reconstruction of the dermis structure so as to accelerate the regeneration of the skin, and has potential application value in the treatment of the full-thickness injury of the skin and the wound surface which is difficult to heal.

In a first aspect, the present invention provides an inorganic-organic composite living cell scaffold. The living cell scaffold comprises a scaffold integral framework formed by constructing inorganic-organic composite biological ink and a plurality of cells which are distributed in a layered manner in the three-dimensional space of the scaffold integral framework; the upper half part of the living cell scaffold is in a layer-by-layer alternating arrangement mode of (A-C) y, wherein A is biological ink, and C is upper-layer cells of the scaffold; the lower half part is in a layer-by-layer alternate arrangement mode of (A-B) x, wherein A is biological ink, and B is cells on the lower layer of the bracket; x and y represent cycle periods which are alternately arranged and are positive integers.

Different from a mode of wrapping cells in hydrogel for printing, the invention adopts a mode of alternately arranging the scaffold and the cells layer by layer, ensures that the cells are tightly adhered to the scaffold layer by layer, exposes the cells to the outside of the hydrogel as far as possible instead of being wrapped by the hydrogel, greatly enhances the contact between the cells and external oxygen nutrients and the timely discharge of metabolic waste, and obviously enhances the interaction of the two cells. Thus, the cells inside the stent show good proliferation, while the direct interaction between fibroblasts and vascular endothelial cells provides the basis for achieving the angiogenic properties of the stent. In addition, the migration of cells in three-dimensional space in the scaffold is more free.

Preferably, the cells on the upper layer of the scaffold are fibroblasts.

Preferably, the cells under the stent are vascular endothelial cells.

The living cell scaffold is characterized in that two cells are layered in the three-dimensional space in the scaffold, and can simulate the cell distribution and physiological structure of the dermis of human skin. Fibroblasts are distributed in the upper half of the scaffold and represent the main cellular constituents of the dermis layer; vascular endothelial cells are distributed in the lower half of the stent, in concert with a dense vascular network at the bottom of the dermal layer. The two cell upper and lower layer distributions used in the present invention are designed to mimic the physiological structure of the natural dermis. An abundant network of blood vessels exists in the dermis of the skin. The vessels in the superficial dermis are small and few, and as the depth increases, the vessels become thicker and denser. One of the important points of the present invention is to produce a bionic dermal substitute, which must structurally simulate the distribution of upper fibroblasts and lower vascular endothelial cells in the natural dermis, so that this arrangement is adopted. When applied to skin lesion repair, this biomimetic alternative works better because of the similar tissue structure that the surrounding tissue retains. However, the above effect cannot be achieved by using the opposite cell distribution manner, i.e., the upper layer cells of the scaffold are vascular endothelial cells, and the lower layer cells of the scaffold are fibroblasts. The scaffold provided by the invention is a functional multi-cell co-culture scaffold, and has a wide application prospect in treatment of skin full-thickness injury and wound surfaces which are difficult to heal.

Preferably, x: y is 3: 7-7: 3. for example, x: y is 3: 7. 4: 6. 5: 5. 6: 4. 7: 3. more preferably, preferably x: y is 1:1. in some embodiments, x is an integer between 3 and 10. In some embodiments, y is an integer between 3 and 10.

The x, y parity placement of the living cell scaffolds of the present invention does not substantially affect the contact interface. Since the circulation of the upper and lower parts of the scaffold starts with a hydrogel layer (layer A in A-B, A-C), a layer of cells (B, C) is distributed over it. When printing is started from bottom to top, the upper cycle of-A-B-A-C-A-C … is started immediately when the lower cycle of A-B-A-B … -A-B ends. This ensures that there must be a layer of hydrogel a between the two cells as a partition, and does not cause the two cells to be simultaneously distributed in one layer. The coexistence of two cells on the same layer should be avoided as much as possible during the preparation process. The fibroblast has relatively stronger proliferation capacity and may influence the survival and proliferation space of vascular endothelial cells on the same layer, so the printing mode adopted in the invention can avoid the situation. Meanwhile, only one layer of hydrogel exists between two cells, so that the two cells are not separated too far on the basis of not being directly mixed and distributed, and the realization of the tight interaction between the cells can be ensured.

Preferably, the inorganic-organic composite bio-ink comprises a bioactive inorganic material and a hydrogel matrix having strength and toughness; the bioactive inorganic material is micron or nanometer particles which release trace elements necessary for human bodies in physiological environment; preferably, the trace elements necessary for human body comprise one or more of Sr, Cu, Co and Si; more preferably, the trace elements essential to the human body include Si and/or Sr.

Preferably, the bioactive inorganic material is SrSiO₃Inorganic fine particles. At this time, the whole framework of the stent is constructed by bio-ink compounded by inorganic particles containing Sr and Si elements and a hydrogel matrix. In the culture process, the composite biological ink releases bioactive Sr and Si ions, acts on two cells in the stent, improves the expression of angiogenesis-related genes and proteins, and achieves the purpose of promoting angiogenesis and accelerating wound repair.

Preferably, the hydrogel matrix is one of gellan gum, hyaluronic acid, sodium alginate, methyl cellulose and collagen; or the hydrogel matrix is a uniform mixture formed by physically combining multiple of gellan gum, hyaluronic acid, sodium alginate, methyl cellulose and collagen.

Preferably, the bioactive inorganic material accounts for 0.7-4% of the mass ratio of the hydrogel matrix. The mass ratio of the bioactive inorganic material to the hydrogel matrix is in the range, inorganic ions released by the bioactive material can be kept at a proper concentration, obvious cytotoxicity cannot be generated, and meanwhile, cells can be stimulated to a certain extent, and the expression of certain genes and proteins in the cells can be improved. In addition, the mechanical property and the forming property of the living cell scaffold are not negatively influenced. Even if inorganic materials are added to the hydrogel matrix at the highest levels, there is no significant negative impact on its mechanical properties.

Preferably, the bioactive inorganic material has a particle size of 20 μm or less. The bioactive inorganic material has regular and uniform appearance, good degradability to release bioactive ions, and no adverse effect on the properties of the hydrogel matrix due to the addition of the inorganic material.

In a second aspect, the present invention provides a method for preparing an inorganic-organic composite living cell scaffold, comprising the steps of:

preparing inorganic-organic composite biological ink;

respectively dissolving cells at the lower layer of the bracket and cells at the upper layer of the bracket in cell compatible liquid to prepare cell suspension 1 and cell suspension 2;

preparing the lower half part of a living cell scaffold which is alternately arranged layer by layer into (A-B) x by using inorganic-organic composite biological ink and cell suspension 1 by using an extrusion biological 3D printing method; similarly, the upper half part of the living cell scaffold which is alternately arranged layer by layer is (A-C) y by using the inorganic-organic composite biological ink and the cell suspension 2 and adopting an extrusion biological 3D printing method; and (4) crosslinking and curing the printed scaffold to obtain the inorganic-organic composite living cell scaffold.

The method is used for preparing the inorganic-organic composite living cell scaffold for the first time, so that the distribution of living cells in a three-dimensional space is accurately controlled, and the adhesion and rapid proliferation of the cells on the scaffold are ensured. The tissue engineering construction body containing living cells and having the capability of simulating the structure of the dermal layer of the skin and regenerating functional tissues is constructed in vitro by the preparation method.

In a third aspect, the present invention provides the use of the inorganic-organic composite living cell scaffold described in any one of the above in the field of skin tissue regeneration, in particular in culturing skin tissue engineering constructs in vitro.

Drawings

FIG. 1 shows SrSiO prepared in the present invention₃Scanning electron micrographs (a, b) and X-ray diffraction analysis (c) of the microparticles;

fig. 2 is a representation of SS-GAM bio-ink prepared in the present invention, including (a) ir spectra of GAM hydrogel and its constituents: (a) GAM, AG, MC, and GG in the above formula refer to sodium alginate/methylcellulose/gellan gum composite hydrogel, sodium alginate, methylcellulose, and gellan gum, respectively; (b) different SrSiO₃Rheology of SS-GAM composite bio-ink with micro-particle content (0%, 2%, 5%, 10%); (c) different SrSiO₃Printing test of SS-GAM composite biological ink with micron particle content (0%, 2%, 5%, 10%);

FIG. 3 shows different SrSiO solid solutions prepared in the present invention₃Characterization of inorganic-organic composite living cell scaffolds with microparticle content (0%, 2%, 5%, 10%): an (a) photomicrograph comprising a stent; (b) scanning electron microscope pictures on the surface; (c) the distribution of Sr element; (d) si element distribution; (e) distribution of O element; (b) (c) the scales in (d) (e) are the same; (f) deformation testing; (g) sr ion release profile; (h) si ion release profile;

FIG. 4 is a fluorescent micrograph of the cell distribution inside an inorganic-organic composite living cell scaffold prepared in the present invention: including (a) day 1 scaffold cross-section; (b) scaffold cross section on day 5; (c) shelf front on day 5;

FIG. 5 shows different SrSiO₃Proliferative activity and angiogenisis gene expression of inorganic-organic composite living cell scaffolds with microparticle content (0%, 2%, 5%, 10%): comprises (a) cell proliferation within 10 days, wherein each group of strip frames sequentially comprises day 1, day 4, day 7 and day 10 from left to right; (b) the first row is a live/dead cell staining micrograph (I, II, III, IV) on day 1, the second row is a live/dead cell staining micrograph (V, VI, VII, VIII) on day 10, and each row is sequentially Co-GAM, Co-2SS-GAM, Co-5SS-GAM and Co-10SS-GAM from left to right; (c) VEGF gene expression; (d) HIF-1 α gene expression; (e) VE-cad gene expression; (f) eNOs-1 gene expression;

FIG. 6 is a drawing showingContaining 0% and 2% SrSiO₃Comparison of microparticle inorganic-organic composite live cell scaffolds with seeded cell scaffolds in (a) proliferative activity, (b) gene expression, and (c) cell adhesion; (a) each group of bar frames comprises GAM printing, GAM inoculating, 2SS-GAM printing and 2SS-GAM inoculating from left to right in sequence; (b) printing 2SS-GAM and inoculating 2SS-GAM in sequence from left to right in each group of strip frames;

FIG. 7 shows the results of the nude mouse subcutaneous transplantation experiment of the inorganic-organic composite living cell scaffold prepared in the present invention: comprising (a) HE staining; (b) CD31 immunohistochemical staining; (c) immunohistochemical staining of human-derived specific CD 31;

FIG. 8 shows the results of the nude mouse wound repair experiment using the inorganic-organic composite living cell scaffold prepared in the present invention: comprises (a) a photo of a nude mouse transplanted with a stent at a wound; (b) photographs of wound healing of nude mice on

days

0, 7, 10, 13, and 15; (c) counting the wound area fraction, wherein each group of bar frames sequentially comprises blank, GAM, 2SS-GAM, GAM + cells and 2SS-GAM + cells from left to right; (d) HE staining of wound site skin on

days

7 and 15; (e) immunohistochemical staining of blood vessels at the wound site with CD 31;

FIG. 9 is a photograph of (a) a diabetic mouse with a scaffold transplanted at a wound site in a diabetic mouse wound repair experiment of an inorganic-organic composite living cell scaffold prepared in the present invention; (b) photographs of wound healing of mice on

days

0, 7, 9, 11, 13, and 15; (b) each group of bar frames is sequentially blank, GAM, 2SS-GAM, GAM + cells and 2SS-GAM + cells from left to right, and the right side of the bar frame is an asterisk which does not substantially influence the numerical value represented by the bar frame; (c) carrying out statistics on wound area fraction; (d) HE staining of wound site skin on day 15; (e) masson trichrome staining of skin at wound sites on day 15;

FIG. 10 is a statistics of the area percentage of collagen fibers in the diabetic mouse wound repair experiment of the inorganic-organic composite living cell scaffold prepared in the present invention, which is blank, GAM, 2SS-GAM, GAM + cells, 2SS-GAM + cells, from left to right.

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.

The invention discloses an inorganic-organic composite living cell scaffold which can simulate the physiological structure of human skin dermis containing blood vessels, help the blood vessels to rebuild and further accelerate the skin regeneration. The scaffold is characterized in that two cells are layered in the three-dimensional space in the scaffold, and the cell distribution and physiological structure of the dermis layer of human skin can be simulated. Fibroblasts are distributed in the upper half of the scaffold and represent the main cellular constituents of the dermis layer; vascular endothelial cells are distributed in the lower half of the stent, in concert with a dense vascular network at the bottom of the dermal layer.

The invention discloses a more refined bionic dermis structure which can simultaneously simulate the physiological structure, cell types and microenvironment of a dermis. Aiming at the physiological structure that the upper part of the dermis layer of the skin of a human body is a collagen fiber network and the lower part of the dermis layer is a dense blood vessel network, the invention prepares the bracket with a regular reticular structure by a biological 3D printing technology, and fibroblasts and blood vessel endothelial cells are densely arranged in an upper-lower layered manner in the structure. In the vertical direction, the layered distribution is similar to the multi-cell distribution of the natural dermis; in the horizontal direction, the network arrangement of cells follows the collagen and vascular network structure in the dermis. The invention focuses on the establishment of a vascular network in a bionic dermis layer structure, and is a more accurate simulation of a dermis physiological structure on a cell scale, which is different from a vascularized dermis structure formed by directly mixing vascular endothelial cells and fibroblasts. In addition, the scaffold of the present invention can be considered as a natural dermal substitute, which has a biomimetic structure highly similar to natural tissue, and is an all-round simulation of cells, structures and microenvironment of human tissue, and which has a macro macroporous structure capable of ensuring sufficient contact of internal cells with oxygen nutrients. More importantly, the structure of the invention is added with functional silicate microparticles besides hydrogel, so as to induce the regeneration of blood vessels and tissues for a long time.

By way of example, the integral framework of the stent is constructed by biological ink compounded by inorganic bioactive materials containing Sr and Si elements and hydrogel. In the culture process, the composite biological ink releases bioactive Sr and Si ions, acts on two cells in the stent, and promotes angiogenesis so as to accelerate wound repair.

The hydrogel in the inorganic-organic composite living cell scaffold is an aqueous solution formed by mixing and dissolving one or more of gellan gum, hyaluronic acid, sodium alginate, methylcellulose and collagen in a certain proportion, has certain formability and can be crosslinked and solidified under certain conditions. Preferably GAM hydrogel material prepared by mixing gellan gum, sodium alginate and methylcellulose in a certain proportion. In some embodiments, the GAM hydrogel comprises 1-10% by weight of gellan gum, sodium alginate and methylcellulose.

The concentration of the gellan gum is 1-4%. The gellan gum is brittle, has high hardness after crosslinking but poor toughness, and therefore the concentration should be maintained within an appropriate range. Preferably, the gellan gum concentration is 2.8%.

The concentration of the sodium alginate is 1-3%. Sodium alginate is soft after crosslinking, can neutralize the defect of poor toughness of gellan gum, but due to the existence of a large amount of sodium ions, the cell is easily adversely affected by too high concentration. Preferably, the sodium alginate concentration is 1.6%.

The concentration of the methyl cellulose is 1-3%. The methyl cellulose aqueous solution can greatly improve the viscosity of the biological ink and improve the forming performance of the biological ink. Preferably, the methylcellulose concentration is 2.8%.

The inorganic bioactive material containing Sr and Si elements in the inorganic-organic composite living cell scaffold is preferably SrSiO₃Micron particles. The SrSiO can be prepared by a hydrothermal method₃And (3) microparticles. Dissolving Sr source and Si source in solvent, slowly dripping Sr source solution into Si source solution, mixing the Sr source solution and the Si source solution, adding alcohol, stirring, hydrothermally heating, washing and drying to obtain SrSiO₃Micron particles. The Sr source is Sr-containing acid, preferably Sr (NO)₃)₂、SrCl₂At least one of (a); the Si source is an acid containing Si, preferably Na₂SiO₃(ii) a The solvent is at least one of deionized water, pure water and ultrapure water; the alcohol is preferably anhydrous ethyl alcoholAn alcohol. In a preferred embodiment provided by the present invention, Sr (NO) is used₃)₂And Na₂SiO₃·9H₂O is used as an Sr source and an Si source and is prepared by a hydrothermal method. The specific method comprises the following steps: respectively weighing a certain amount of Sr (NO)₃)₂Crystals and Na₂SiO₃·9H₂And O, adding equal amount of deionized water to completely dissolve the two. Sr (NO)₃)₂The solution is slowly added with Na₂SiO₃The solution was added dropwise with stirring until the two were completely mixed. Adding a certain amount of anhydrous ethanol, and stirring for more than 30 min. Then the mixed solution is poured into a 50ml polytetrafluoroethylene hydrothermal reaction kettle, and hydrothermal reaction is carried out for 8 hours at 160 ℃. After the solution is cooled, the supernatant is poured off, the lower white precipitate is collected, and the SrSiO is obtained after deionized water washing, alcohol washing and drying₃Micron particles. Sr (NO) weighed as above₃)₂And Na₂SiO₃·9H₂Molar ratio of O crystal is 1:1, Sr (NO)₃)₂Solution and Na₂SiO₃The concentration of the solution is 0.2mol/L, Sr (NO)₃)₂Solution and Na₂SiO₃The volume ratio of the solution to the absolute ethyl alcohol is 1:1: 1.5.

The invention prepares SrSiO by the method for the first time₃Micron particles are obtained to obtain the near hexagonal prism-shaped SrSiO with regular appearance₃Microparticles having high purity and high crystallinity. Simultaneously explores the bioactivity of the SrSiO and finds that the prepared SrSiO₃The micrometer granule has effect in promoting angiogenesis gene expression of co-cultured cells. SrSiO in the invention₃The microparticles act as a replacement for growth factors to produce cell induction. The common method of inducing vascularization is to add growth factors thereto, but this method is costly and growth factors are difficult to maintain for a long time. Based on the situation, the invention selects SrSiO₃The microparticles are used as bioactive factors capable of stably and continuously exerting cell induction to replace the traditional protein growth factors to prepare the skin substitute scaffold with long-term vascularization capacity.

Sr element and Si element are proved to have the capacity of promoting angiogenesis, and Sr can obviously improve the expression of the angiogenesis factor VEGF in cells. In addition, Si element can also accelerate collagen deposition and wound repair during the wound healing process. The invention considers that the two elements are introduced into the stent, Sr and Si ions are gradually released through degradation, and a continuous stimulation effect is generated on printing, so that the blood vessel forming activity of the stent is improved, and the skin regeneration is promoted.

It is worth to say that the application of Sr-Si biomaterials in skin wound repair is rarely explored in the field of tissue engineering, and most of the research on Sr element is focused in the field of bone repair. The invention creatively selects SrSiO₃The material is applied to skin tissue regeneration, and the preparation method is optimized to obtain hexagonal prism-shaped SrSiO with special appearance₃And (3) microparticles. Good result feedback is obtained after the skin substitute stent is applied to the skin substitute stent.

The invention also provides a preparation method of the inorganic-organic composite biological ink. Dissolving gellan gum powder in solvent at 90 deg.C, stirring to obtain gellan gum solution, adding SrSiO₃And stirring and cooling the particles, the sodium alginate and the methylcellulose powder to obtain the SS-GAM inorganic-organic composite biological ink. The solvent is at least one of deionized water, pure water and ultrapure water.

The invention uses the method to prepare the SS-GAM inorganic-organic composite biological ink for the first time to obtain SrSiO₃The composite material has the advantages that the microparticles are uniformly distributed in the hydrogel, the biological ink has good printing performance and formability, and can slowly release bioactive ions so as to generate positive effects on cell activities.

SrSiO in the composite biological ink of the inorganic-organic composite living cell scaffold₃The content of the micron particles is 0-10% of the mass of the gellan gum powder in the hydrogel. Too high SrSiO₃The microparticle content affects the printability of the bio-ink and higher concentrations of Sr, Si ions are released, possibly adversely affecting the cells.

The volume ratio of the two cell suspensions in the inorganic-organic composite living cell scaffold is 1:1, the concentration ratio of the two cells is 1:1, and the concentration range is 3000-. Too high concentration of cell suspension can cause too high cell density on the scaffold and influence cell adhesion and proliferation; too low a concentration of the cell suspension may result in too low a cell density and thus affect the activity of the cells. Preferably the concentration of the cell suspension is 10000/μ L.

The inorganic-organic composite living cell scaffold is prepared layer by adopting an extrusion type biological 3D printing method. The lower half part of the stent is in a layer-by-layer alternating arrangement mode of (A-B) x, wherein A is an inorganic-organic composite biological ink material, and B is cells on the lower layer of the stent, in particular vascular endothelial cells. The upper half part of the scaffold is in a layer-by-layer alternating arrangement mode of (A-C) y, wherein C is cells on the upper layer of the scaffold, and specifically fibroblasts. And x and y represent a cycle period and are positive integers, and the ratio of x to y is 1:1.

The following shows a specific preparation method of the multicellular biological composite scaffold in one embodiment of the invention:

dissolving a certain amount of gellan gum powder in deionized water, and (C) at high temperature>Stirring at 90 deg.C for more than 30min until the powder is completely dissolved. And closing the heating switch to naturally cool the heating switch. When the gellan gum temperature is reduced to below 84 ℃, weighing a certain amount of high-viscosity sodium alginate, methylcellulose and SrSiO₃The microparticles are added and mixed thoroughly with continuous stirring. When the temperature of the completely mixed high-viscosity fluid is reduced to room temperature, a soft slurry similar to a solid state is formed, namely the SS-GAM inorganic-organic composite biological ink. The concentration of the gellan gum is 1-4%, and the preferred concentration is 2.8%. The concentration of the sodium alginate is 1-3%, and the preferred concentration is 1.6%. The concentration of the methyl cellulose is 1-3%, and the preferred concentration is 2.8%. SrSiO₃The content of the micron particles is 0-10% of the mass of the gellan gum powder in the hydrogel. Note that all SrSiO described in the present invention₃The content of the micron particles in the SS-GAM inorganic-organic composite biological ink is the percentage of the mass of the micron particles relative to the mass of the gellan gum powder.

Cell suspension preparation: preparing vascular endothelial cells, and dispersing the vascular endothelial cells in a cell-compatible liquid to prepare a cell suspension 1; fibroblasts are prepared and dispersed in a cell-compatible liquid to prepare a cell suspension 2. The cell-compatible liquid is at least one of endothelial cell culture medium ECM and high-sugar DMEM culture medium. Endothelial cell culture media ECM is preferred because of the higher culture environment requirements for both cells compared to vascular endothelial cells. The cell concentration in the cell suspension is in the range of 3000-30000 cells/μ L, preferably 10000 cells/μ L.

Biological 3D printing process: printer start and print program settings. The preparation process involves two printing modules, including an air pressure push extrusion type needle and a micro piezoelectric sample application needle, which are respectively used as printing extrusion channels of composite bio-ink and cell suspension. Pushing an extrusion type needle head to extrude the composite biological ink A by air pressure, absorbing the cell suspension 1 by using a miniature piezoelectric sample application needle head, spraying the cell suspension on the biological ink on the lower layer, and circulating in such a way to obtain a lower half part of the stent which presents a layer-by-layer alternate arrangement mode of (A-B) × x, wherein B is vascular endothelial cells; and then, extruding the composite bio-ink by pushing an extrusion type needle head through air pressure, sucking the cell suspension 2 by using a miniature piezoelectric sample application needle head, spraying the cell suspension on the bio-ink on the lower layer, and circulating the steps to obtain the upper half part of the scaffold which presents an (A-C) y layer-by-layer alternate arrangement mode, wherein C is a fibroblast. The stent used after printing contains a certain amount of Ca²⁺The ionic ECM medium is cross-linked and solidified. The control of parameters such as extrusion rate in the printing process is not the innovation of the invention, and the control can be adjusted according to the requirements in the test process. For example, the extrusion pressure of bio-ink A during printing is 230-280kPa, and the frequency of the spotting needle spraying

cell suspensions

1 and 2 is 110 Hz.

The crosslinked and cured scaffolds were placed in a six-well plate. 2mL of ECM medium was added to each scaffold, and the plates were placed in a 37 ℃ incubator for in vitro culture with alternate media.

In the inorganic-organic composite living cell scaffold prepared in the disclosure, two kinds of cells are layered in three-dimensional space in the scaffold, and can simulate the physiological structure of human skin dermis containing blood vessels, and promote the regeneration of blood vessels and the reconstruction of dermis structure.

In some embodiments, the scaffold of the present invention has a porous structure. The porous structure mainly refers to a triangular macroporous structure of the stent in macroscopic view. First, unlike most of the current artificial tissues in the form of continuous sheets or blocks, the scaffold having a mesh-like macroporous structure disclosed in the present invention can increase the air permeability of the scaffold after transplantation as much as possible, ensure that the cells inside the scaffold are fully contacted with oxygen and nutrients, and provide a wide space for cell activities. Secondly, after transplantation, the porous structure has very remarkable promoting effect on the growth of surrounding tissues and blood vessels, and can greatly enhance the combining capacity of the stent and a host, including effective connection of the blood vessels and tissue integration. Finally, the macroscopic macroporous structure can ensure the elasticity and softness of the stent, and the pores deform when the stent is stretched and folded, so that the stent cannot be structurally damaged.

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

SrSiO₃Preparation of microparticles:

weighing 2.116g Sr (NO)₃)₂And 2.842g Na₂SiO₃·9H₂O, respectively measuring 50mL of deionized water in the two beakers, adding the deionized water into the two beakers, and stirring until the crystals are completely dissolved; sr (NO) is added by using a dropper₃)₂The solution is slowly added with Na₂SiO₃Dripping and stirring the solution until the solution and the solution are completely mixed; weighing about 50mL of absolute ethyl alcohol, pouring the absolute ethyl alcohol into the cup, sealing the cup mouth, and stirring for more than 30 min; pouring the mixture into a 50mL polytetrafluoroethylene hydrothermal reaction kettle, putting the kettle into an oven, and carrying out hydrothermal reaction at 160 ℃ for 8 hours(ii) a After the hydrothermal process is finished, pouring out the supernatant after the solution is cooled, collecting the lower white precipitate, washing with deionized water for 3 times, performing ultrasonic treatment for 10min each time, and washing with absolute ethyl alcohol for 3 times, performing ultrasonic treatment for 10min each time; putting the powder into a 60 ℃ oven, drying and grinding to obtain SrSiO₃Micron particles.

FIG. 1 shows SrSiO prepared by the above method₃Scanning electron microscope photograph and X-ray diffraction analysis result of micron particles prove that the prepared pure phase SrSiO with regular shape similar to hexagonal prism₃Micron particles.

Example 2

Preparation of SS-GAM inorganic-organic composite biological ink

Weighing 0.7g of gellan gum powder in a wide-mouth bottle, adding 25mL of sterilized deionized water, placing in a water bath kettle, and stirring at 90 ℃ for more than 30min until the powder is completely dissolved; closing the heating switch to naturally cool the solution; when the temperature of the solution is reduced to be lower than 84 ℃, 0.4g of high-viscosity sodium alginate, 0.7g of methylcellulose and a certain amount (four contents of 0g, 0.014g, 0.035g and 0.07g) of SrSiO are weighed₃Adding the micron particles into the mixture, and continuously stirring the mixture to fully mix the mixture; cooling the high viscosity fluid to room temperature, and irradiating with ultraviolet light for about 1 hr to obtain sterile four gradient SrSiO of GAM, 2SS-GAM, 5SS-GAM, and 10SS-GAM₃Inorganic-organic composite bio-ink of content.

FIG. 2 shows that GAM hydrogel is a product of physical combination of gellan gum, sodium alginate and methylcellulose (see a in FIG. 2), and is compounded with SrSiO₃The micron-sized bio-ink has shear thinning performance (see b in figure 2) required by printing and good forming performance (see c in figure 2), and can be widely applied to preparation of scaffolds with different shapes.

Example 3

Preparation of SS-GAM inorganic-organic composite living cell scaffold

The method comprises the following steps: preparation of gradient SS-GAM inorganic-organic composite biological ink

Weighing 0.7g gellan gum powder in a wide-mouth bottle, adding 25mL deionized water, placing in a water bath kettle, stirring at 90 deg.C for more than 30min until the powder is completely dissolvedSolving; closing the heating switch to naturally cool the solution; when the temperature of the solution is reduced to be lower than 84 ℃, 0.4g of high-viscosity sodium alginate, 0.7g of methylcellulose and a certain amount (four gradient contents of 0g, 0.014g, 0.035g and 0.07g) of SrSiO are weighed₃Adding the micron particles into the mixture, and continuously stirring the mixture to fully mix the mixture; cooling the high viscosity fluid to room temperature, and irradiating with ultraviolet light for about 1 hr to obtain sterile four gradient SrSiO of GAM, 2SS-GAM, 5SS-GAM, and 10SS-GAM₃Inorganic-organic composite bio-ink of content.

Step two: preparation of

cell suspensions

1, 2

Uniformly dispersing the vascular endothelial cells in culture in an endothelial cell culture medium ECM, wherein the cell density is 10000/mu L, and obtaining a cell suspension 1; and (3) uniformly dispersing the cultured fibroblasts in an endothelial cell culture medium ECM, wherein the cell density is 10000/mu L, and thus obtaining a cell suspension 2.

Step three: printing SS-GAM inorganic-organic composite living cell scaffold

a. A printing program is set. The printing support is of a regular hexagonal porous structure, and the shape of the pores is triangular. The radius of the bracket is 8mm, and the thickness is about 1.5 mm.

b. Cleaning the miniature piezoelectric sample application needle with sterile water for several times, wiping for later use, filling the prepared SS-GAM inorganic-organic composite bio-ink into a charging barrel, and loading into an air pressure pushing extrusion type printing module; the

prepared cell suspensions

1 and 2 are respectively added into the

wells

1 and 2 of a 96-well plate for standby.

c. Pushing an extrusion type needle head to extrude the composite biological ink by air pressure, absorbing the cell suspension 1 by using a miniature piezoelectric sample application needle head, spraying the cell suspension on the biological ink on the lower layer, and circulating in such a way to obtain a lower half part of the stent which presents a layer-by-layer alternating arrangement mode of (A-B) × x, wherein B is vascular endothelial cells; and then, extruding the composite bio-ink by pushing an extrusion type needle head through air pressure, sucking the cell suspension 2 by using a miniature piezoelectric sample application needle head, spraying the cell suspension on the upper bio-ink layer, and circulating the steps to obtain the upper half part of the scaffold which presents an (A-C) y layer-by-layer alternate arrangement mode, wherein C is a fibroblast. In this embodiment, x is 5, y is 5, and x: y is 1:1.

Four gradient SrSiO of GAM, 2SS-GAM, 5SS-GAM and 10SS-GAM are used₃The inorganic-organic composite biological ink with the content is respectively printed. The extrusion pressure range of the biological ink in the printing process is 230-280kPa, and the frequency of the spotting needle spraying the

cell suspensions

1 and 2 is 110 Hz. Finally, four SS-GAM inorganic-organic composite living cell scaffolds are obtained, and are named as Co-GAM, Co-2SS-GAM, Co-5SS-GAM and Co-10SS-GAM respectively.

The stent used after printing contains a certain amount of Ca²⁺The ionic ECM medium is cross-linked and solidified. The crosslinked and cured scaffolds were placed in a six-well plate. 2mL of ECM medium was added to each scaffold, and the plates were placed in a 37 ℃ incubator for in vitro culture with alternate media.

Fig. 3 shows the printed shape of four stents, which are flexible and can be folded three times in succession without shape failure. SrSiO₃The micron particles are uniformly dispersed in the stent without agglomeration. Due to SrSiO₃The four scaffolds can release Sr and Si ions with different concentrations in a gradient way without using the content of micron particles.

Example 4

Cell distribution of SS-GAM inorganic-organic composite living cell scaffolds

The method comprises the following steps: preparation of SS-GAM inorganic-organic composite biological ink

Weighing 0.7g of gellan gum powder into a wide-mouth bottle, adding 25mL of deionized water, placing the wide-mouth bottle in a water bath kettle, and stirring for more than 30min at 90 ℃ until the powder is completely dissolved; closing the heating switch to naturally cool the solution; when the temperature of the solution is reduced to below 84 ℃, 0.4g of high-viscosity sodium alginate, 0.7g of methylcellulose and a certain amount (0g-0.07g) of SrSiO are weighed₃Adding the micron particles into the mixture, and continuously stirring the mixture to fully mix the mixture; and (3) cooling the temperature of the completely mixed high-viscosity fluid to room temperature, and irradiating for about 1h under ultraviolet light to obtain the sterile SS-GAM inorganic-organic composite biological ink.

Step two: preparation of fluorescently labeled

cell suspensions

1, 2

Marking the vascular endothelial cells in culture with green fluorescence, uniformly dispersing the vascular endothelial cells in an endothelial cell culture medium ECM, wherein the cell density is 10000/mu L, and obtaining a cell suspension 1 (containing green fluorescence); the fibroblasts in culture are uniformly marked with red fluorescence and dispersed in an endothelial cell culture medium ECM, and the cell density is 10000 cells/mu L, so that a cell suspension 2 (containing red fluorescence) is obtained.

Step three: printing SS-GAM inorganic-organic composite living cell scaffold

b. Cleaning the micro piezoelectric sample application needle with sterile water for several times, wiping for later use, filling the prepared SS-GAM inorganic-organic composite bio-ink into a charging barrel, and loading into an air pressure pushing extrusion type printing module; the

prepared cell suspensions

1 and 2 are respectively added into the

wells

1 and 2 of a 96-well plate for standby.

c. Pushing an extrusion type needle head to extrude the composite biological ink by air pressure, absorbing the cell suspension 1 by using a miniature piezoelectric sample application needle head, spraying the cell suspension on the biological ink on the lower layer, and circulating in such a way to obtain a lower half part of the stent which presents a layer-by-layer alternating arrangement mode of (A-B) × x, wherein B is vascular endothelial cells; and then, extruding the composite bio-ink by pushing an extrusion type needle head through air pressure, sucking the cell suspension 2 by using a miniature piezoelectric sample application needle head, spraying the cell suspension on the upper bio-ink layer, and circulating the steps to obtain the upper half part of the scaffold which presents an (A-C) y layer-by-layer alternate arrangement mode, wherein C is a fibroblast. The extrusion pressure range of the biological ink in the printing process is 230-280kPa, and the frequency of the spotting needle spraying the

cell suspensions

1 and 2 is 110 Hz. Finally obtaining the SS-GAM inorganic-organic composite living cell scaffold. In this embodiment, x is 5, y is 5, and x: y is 1:1.

d. The stent used after printing contains a certain amount of Ca²⁺The ionic ECM medium is cross-linked and solidified. The crosslinked and cured scaffolds were placed in a six-well plate. 2mL of ECM medium was added to each scaffold, and the plates were placed in a 37 ℃ incubator for in vitro culture with alternate media.

e. The scaffold was cut from the middle and the distribution of both cells at the same cross section was observed using a microscope.

FIG. 4 shows that vascular endothelial cells with green fluorescence are distributed in the lower layer of the stent and fibroblasts with red fluorescence are distributed in the upper layer of the stent. This distribution can be observed from both the cross-section and the front side and can be maintained at least until day 5. This again demonstrates the cellular hierarchy inside the SS-GAM inorganic-organic composite living cell scaffold.

Example 5

Cell viability of SS-GAM inorganic-organic composite viable cell scaffolds

Weighing 0.7g of gellan gum powder into a wide-mouth bottle, adding 25mL of deionized water, placing the wide-mouth bottle in a water bath kettle, and stirring for more than 30min at 90 ℃ until the powder is completely dissolved; closing the heating switch to naturally cool the solution; when the temperature of the solution is reduced to be lower than 84 ℃, 0.4g of high-viscosity sodium alginate, 0.7g of methylcellulose and a certain amount (four gradient contents of 0g, 0.014g, 0.035g and 0.07g) of SrSiO are weighed₃Adding the micron particles into the mixture, and continuously stirring the mixture to fully mix the mixture; cooling the high viscosity fluid to room temperature, and irradiating with ultraviolet light for about 1 hr to obtain sterile four gradient SrSiO of GAM, 2SS-GAM, 5SS-GAM, and 10SS-GAM₃Inorganic-organic composite bio-ink of content.

Step two: preparation of

cell suspensions

1, 2

Step three: printing SS-GAM inorganic-organic composite living cell scaffold

prepared cell suspensions

1 and 2 are respectively added into the

wells

1 and 2 of a 96-well plate for standby.

d. Four gradient SrSiO of GAM, 2SS-GAM, 5SS-GAM and 10SS-GAM are used₃The inorganic-organic composite biological ink with the content is respectively printed. The extrusion pressure range of the biological ink in the printing process is 230-280kPa, and the frequency of the spotting needle spraying the

cell suspensions

e. The stent used after printing contains a certain amount of Ca²⁺The ionic ECM medium is cross-linked and solidified. The crosslinked and cured scaffolds were placed in a six-well plate. 2mL of ECM medium was added to each scaffold, and the plates were placed in a 37 ℃ incubator for in vitro culture with alternate media.

The proliferation activity of cells on the scaffolds was measured using the CCk-8 kit on

days

1,4,7, 10. A and b in FIG. 5 indicate that the cells on the scaffold were able to maintain good proliferative activity for a long period of time, the cell number showed a steady increase, and three groups except the Co-10SS-GAM group at day 10 were able to support cell proliferation while maintaining the shape of the scaffold. From the results of cell proliferation alone, the cells in the four scaffolds were able to maintain good proliferation. However, as can be seen from the fluorescence micrographs, although the number of cells in the Co-10SS-GAM group is increased, the scaffold itself becomes scattered and loses the regular macroporous morphology, and it is obvious that in this case, the structural stability of the scaffold is relatively low (the influence caused by the high content of SS), and the scaffold cannot support the long-term activity of the cells on the basis of maintaining the self-triangular pore structure, so that the application of the scaffold in transplantation is not facilitated.

And (3) characterizing the expression condition of the angiogenesis-related genes of the cells in the scaffold by using an RT-PCR method. Specifically, after the scaffold is cultured for 5 days, total RNA of each group of cells is extracted by a Trizol method, RNA is reversely transcribed into cDNA by a ReverTra Ace-alpha kit, and the gene expression conditions of the two cells are explored by adopting a SYBR Green fluorescent real-time quantitative PCR method. As can be seen from the RT-PCR results, a certain amount of SrSiO₃The micron particles can play a certain role in promoting the expression of the angiogenesis-related genes of the stent cells. In combination, Co-2SS-GAM was able to simultaneously promote the expression of four angiogenesis-related genes (see c-f in FIG. 5) compared to the control group. Thus, we prefer 2% SrSiO₃Co-2SS-GAM composite scaffold with micron particle content.

The results show that the prepared SS-GAM inorganic-organic composite living cell scaffold has high biological activity and angiogenesis characteristic, and has great application potential in the aspects of promoting the reconstruction of a dermis structure and accelerating the wound repair.

Example 6

Comparison of the Performance of the inorganic-organic composite Living cell scaffolds with scaffolds prepared by cell inoculation

The method comprises the following steps: preparation of 2SS-GAM inorganic-organic composite biological ink

Weighing 0.7g of gellan gum powder into a wide-mouth bottle, adding 25mL of deionized water, placing the wide-mouth bottle in a water bath kettle, and stirring for more than 30min at 90 ℃ until the powder is completely dissolved; closing the heating switch to naturally cool the solution; when the temperature of the solution is reduced to below 84 ℃, 0.4g of high-viscosity sodium alginate, 0.7g of methylcellulose and 0.014g of SrSiO are weighed₃Adding the micron particles into the mixture, and continuously stirring the mixture to fully mix the mixture; and (3) cooling the temperature of the completely mixed high-viscosity fluid to room temperature, and irradiating for about 1h under ultraviolet light to obtain the sterile 2SS-GAM inorganic-organic composite biological ink.

Step two: preparation of

cell suspensions

1, 2

Step three: printing SS-GAM inorganic-organic composite living cell scaffold

b. Cleaning the micro piezoelectric sample application needle with sterile water for several times, wiping for later use, filling the prepared 2SS-GAM inorganic-organic composite bio-ink into a charging barrel, and filling the charging barrel into an air pressure pushing extrusion type printing module; the

prepared cell suspensions

1 and 2 are respectively added into the

wells

1 and 2 of a 96-well plate for standby.

d. The extrusion pressure range of the biological ink in the printing process is 230-240kPa, and the frequency of the spotting needle spraying the

cell suspensions

1 and 2 is 110 Hz. Finally obtaining the Co-2SS-GAM viable cell scaffold for biological printing.

Step four: scaffolds prepared by cell-plating method as control group

b. The extrusion type needle head is pushed by air pressure to extrude the 2SS-GAM composite biological ink, the extrusion pressure range of the biological ink in the printing process is 230-240kPa, and the 2SS-GAM pure material bracket is finally obtained.

c. Taking the cell suspension 1 and the cell suspension 2 with the same volume, and sequentially inoculating the cell suspension 1 and the cell suspension 2 on the 2SS-GAM pure material bracket by using a pipette gun. Obtaining the Co-2SS-GAM bracket inoculated by the cells.

d. Using the inoculated stent containing a certain amount of Ca²⁺The ionic ECM medium is cross-linked and solidified. The crosslinked and cured scaffolds were placed in a six-well plate. 2mL of ECM medium was added to each scaffold, and the plates were placed in a 37 ℃ incubator for in vitro culture with alternate media.

After 5 days of in vitro culture, the biological 3D printing technique is significantly superior from the results of cell proliferation and angiogenesis-related gene expression on both sets of scaffolds (see a, b in fig. 6). It can be seen visually from the photographs of cell distribution (green fluorescence) on the scaffold after 1,4,7 days of culture taken by confocal microscopy, that the biological 3D printing method can achieve a large amount of uniform and dispersed distribution of cells on the scaffold, whereas the conventional cell seeding method can result in very low cell adhesion and uneven distribution (see c in fig. 6).

The results show that the biological 3D printing method adopted by the people can provide space for cell proliferation and gene expression on the basis of controlling cell distribution and enhancing cell adhesion. Since the main body of the stent is made of hydrogel material, the hydrogel material with rich water content usually presents a smoother surface appearance, which is not very beneficial to cell adhesion. Therefore, when the traditional method for inoculating cells on the pure material scaffold for 3D printing is adopted, most of the cells are deposited at the bottom of the pore plate under the action of gravity due to the fact that the designed scaffold has a mesh macroporous structure, and the cells can only depend on the weak attachment capacity of the cells to hydrogel, so that the conditions that the cell adhesion amount on the surface of the scaffold is very small and very uneven are caused; the cell writing method adopted by the invention uses a piezoelectric precision needle to spray cell suspension during printing, positions the cell suspension accurately on the central axis of each hydrogel material, and sprays fine liquid drops, so that the adhesion rate of cells on the smooth surface of the hydrogel can be greatly improved, and the high survival rate of the cells is ensured.

Example 7

Subcutaneous transplantation experiment of inorganic-organic composite living cell scaffold

The method comprises the following steps: preparation of GAM and 2SS-GAM inorganic-organic composite biological ink

Weighing 0.7g of gellan gum powder into a wide-mouth bottle, adding 25mL of deionized water, placing the wide-mouth bottle in a water bath kettle, and stirring for more than 30min at 90 ℃ until the powder is completely dissolved; closing the heating switch to naturally cool the solution; when the temperature of the solution is reduced to below 84 ℃, 0.4g of high-viscosity sodium alginate, 0.7g of methylcellulose and a certain amount (0g, 0.014g of two contents) of SrSiO are weighed₃Adding the micron particles into the mixture, and continuously stirring the mixture to fully mix the mixture; cooling the high viscosity fluid to room temperature, and irradiating under ultraviolet light for about 1 hr to obtain sterile GAM and 2SS-GAM inorganic-organic composite biological ink.

Step two: preparation of

cell suspensions

1, 2

Step three: printing SS-GAM inorganic-organic composite living cell scaffold

a. A printing program is set. The printing support is of a regular hexagonal porous structure, and the shape of the pores is triangular. The radius of the bracket is 5mm, and the thickness is about 2 mm.

prepared cell suspensions

1 and 2 are respectively added into the

wells

1 and 2 of a 96-well plate for standby.

c. Pushing an extrusion type needle head to extrude the composite biological ink by air pressure, absorbing the cell suspension 1 by using a miniature piezoelectric sample application needle head, spraying the cell suspension on the biological ink on the lower layer, and circulating in such a way to obtain a lower half part of the stent which presents a layer-by-layer alternating arrangement mode of (A-B) × x, wherein B is vascular endothelial cells; and then, extruding the composite bio-ink by pushing an extrusion type needle head through air pressure, sucking the cell suspension 2 by using a miniature piezoelectric sample application needle head, spraying the cell suspension on the upper bio-ink layer, and circulating the steps to obtain the upper half part of the scaffold which presents an (A-C) y layer-by-layer alternate arrangement mode, wherein C is a fibroblast. In this embodiment, x is 10, y is 10, and x is 1:1.

d. And respectively printing with GAM and 2SS-GAM inorganic-organic composite biological ink. The extrusion pressure range of the biological ink in the printing process is 230-250kPa, and the frequency of the spotting needle spraying the

cell suspensions

1 and 2 is 110 Hz. Finally obtaining 2SS-GAM inorganic-organic composite living cell scaffolds which are named as GAM + cells and 2SS-GAM + cells respectively.

e. Printing pure material scaffolds with GAM and 2SS-GAM inorganic-organic composite bio-ink, respectively, with the same shape, size and thickness as the above living cell scaffolds. The printed scaffolds were named GAM and 2SS-GAM scaffolds.

f. The stent used after printing contains a certain amount of Ca²⁺The ionic ECM medium is cross-linked and solidified. The crosslinked cured scaffolds were placed in a twelve well plate. 1mL of ECM medium was added to each scaffold, and the plates were placed in a 37 ℃ incubator for in vitro culture with alternate media.

Step four: subcutaneous implantation of stents

Male BALB/c nude mice (SPF clean grade) were selected at 8 weeks of age. After a nude mouse is anesthetized by intraperitoneal injection of pentobarbital sodium, a cotton ball is dipped in iodophor to sterilize back skin, two longitudinal incisions are respectively made on two sides of a midline of the back, and subcutaneous fascia is bluntly separated by using ophthalmological forceps to form a cavity. The four scaffolds were cut in half, with half of the scaffold being a sample. Two different groups of samples were implanted into each nude mouse, the incision was sutured with sterile surgical thread and sterilized with iodophor, and the nude mice were housed in divided cages in SPF environment. The material was taken four weeks after transplantation and immunohistochemical analysis was performed on the samples.

From the HE staining results, the amount of cells inside the live cell scaffold sample was much larger than the pure material scaffold group (see a in fig. 7). The CD31 immunohistochemical staining results showed the number of blood vessels in the sample (see b in fig. 7). The sample was specifically stained with human CD31 to distinguish human vascular endothelial cells printed on the stent from nude mouse autologous vascular endothelial cells. The former two groups of unprinted cells do not generate color reaction, and the latter two groups of unprinted cells have human blood vessels, which proves that the biological 3D printed living cell scaffold can spontaneously form blood vessels in an in-vivo environment on the basis of ensuring that scaffold cells survive for a long time, and the blood vessels have blood cells, so that the fusion of the biological 3D printed living cell scaffold and a host vascular system is proved. The presence of a greater number of human blood vessels in the 2SS-GAM + cells group samples than in the GAM + cells group demonstrated that SrSiO₃The microparticles contribute to the vascular effect (see c in fig. 7).

The results show that the prepared 2SS-GAM inorganic-organic composite living cell scaffold has high biological activity and angiogenesis characteristics in an in vivo environment, can ensure the long-term survival of cells in the scaffold and support endothelial cells to form a functional vascular structure.

Example 8

Nude mouse wound repair experiment of inorganic-organic composite living cell scaffold

Step two: preparation of

cell suspensions

1, 2

Step three: printing SS-GAM inorganic-organic composite living cell scaffold

a. A printing program is set. The printing support is of a regular hexagonal porous structure, and the shape of the pores is triangular. The radius of the bracket is 7.5mm, and the thickness is about 1 mm.

prepared cell suspensions

1 and 2 are respectively added into the

wells

1 and 2 of a 96-well plate for standby.

c. Pushing an extrusion type needle head to extrude the composite biological ink by air pressure, absorbing the cell suspension 1 by using a miniature piezoelectric sample application needle head, spraying the cell suspension on the biological ink on the lower layer, and circulating in such a way to obtain a lower half part of the stent which presents a layer-by-layer alternating arrangement mode of (A-B) × x, wherein B is vascular endothelial cells; and then, extruding the composite bio-ink by pushing an extrusion type needle head through air pressure, sucking the cell suspension 2 by using a miniature piezoelectric sample application needle head, spraying the cell suspension on the upper bio-ink layer, and circulating the steps to obtain the upper half part of the scaffold which presents an (A-C) y layer-by-layer alternate arrangement mode, wherein C is a fibroblast. In this embodiment, x is 3, y is 3, and x: y is 1:1.

d. Printing with GAM and 2SS-GAM inorganic-organic composite biological ink respectively. The extrusion pressure range of the biological ink in the printing process is 230-250kPa, and the frequency of the spotting needle spraying the

cell suspensions

e. Printing pure material scaffolds with the same shape, size and thickness as the above living cell scaffolds using GAM and 2SS-GAM inorganic-organic composite bio-ink, respectively. The printed scaffolds were named GAM and 2SS-GAM scaffolds.

Step four: nude mouse wound surface transplantation of scaffold

Male BALB/c nude mice (SPF clean grade) 6-8 weeks old were selected. After nude mice were anesthetized by intraperitoneal injection of pentobarbital sodium, the cotton balls were dipped in iodophor to sterilize the back skin, and a circular full-thickness skin defect wound surface with a diameter of 15mm was made on the back and randomly divided into 5 groups. The stent was applied to the wound site and secured with gauze and medical transparent dressing (Tegaderm TM, 3M). Nude mice were housed in divided cages in an SPF environment. Wound healing was observed and histological analysis of new skin was performed.

From the results, it can be seen that the 2SS-GAM, GAM + cells and 2SS-GAM + cells scaffolds can accelerate the wound healing rate compared to the Blank group and the pure hydrogel GAM group (see a, b in FIG. 8). HE staining at 7 days showed a scaffold (black dashed line) transplanted at the wound site, and after 15 days three groups had formed intact epithelium and the wound healed well (see d in fig. 8). The 2SS-GAM + cells group had the most angiogenesis in the wound site CD31 staining results, followed by the 2SS-GAM group, indicating that SS has a promoting effect on angiogenesis in the wound healing process (see e in fig. 8).

The above results indicate that SrSiO is present in vivo₃The micrometer particles have positive effect on blood vessel regeneration, and the prepared living cell scaffold can accelerate wound healing and has great application potential in the aspect of skin regeneration.

Example 9

Diabetes mouse wound repair experiment of inorganic-organic composite living cell scaffold

Weighing 0.7g of gellan gum powder into a wide-mouth bottle, adding 25mL of deionized water, placing the wide-mouth bottle in a water bath kettle, and stirring for more than 30min at 90 ℃ until the powder is completely dissolved; close and addA thermal switch is used for naturally cooling the solution; when the temperature of the solution is reduced to below 84 ℃, 0.4g of high-viscosity sodium alginate, 0.7g of methylcellulose and a certain amount (0g, 0.014g of two contents) of SrSiO are weighed₃Adding the micron particles into the mixture, and continuously stirring the mixture to fully mix the mixture; cooling the high viscosity fluid to room temperature, and irradiating under ultraviolet light for about 1 hr to obtain sterile GAM and 2SS-GAM inorganic-organic composite biological ink.

Step two: preparation of

cell suspensions

1, 2

Step three: printing SS-GAM inorganic-organic composite living cell scaffold

prepared cell suspensions

1 and 2 are respectively added into the

wells

1 and 2 of a 96-well plate for standby.

cell suspensions

Step four: diabetic wound grafting of stents

a. Male C57BL/6 mice 7-8 weeks old were selected, and the mouse diabetes model was induced by intraperitoneal injection of Streptozotocin (STZ) as follows: dissolving STZ in 0.1mol/L sodium citrate buffer solution (pH 4.5), preparing for use, and operating on ice in a dark place; subcutaneously injecting 50mg/kg of the injection into the abdominal cavity of a male C57BL/6 laboratory mouse (7-8 weeks), injecting 1 time every 2 days, and continuously injecting for 10 days; and thirdly, measuring the blood sugar after 4 weeks, wherein the rising value of the blood sugar is higher than 20mmol/L after 1 week, and the mouse has the characteristics of polydipsia, polyphagia, diuresis and weight loss, which indicates that the mouse is successfully induced into a diabetes model and can be used for subsequent experiments.

b. After removing hair from the dorsal area of the mice, a circular full-thickness skin defect wound of 15mm in diameter was created and randomly divided into 5 groups. The stent was applied to the wound site and secured with gauze and medical transparent dressing (Tegaderm TM, 3M). Diabetic mice were housed in divided cages in an SPF environment. Wound healing was observed and histological analysis of new skin was performed.

As can be seen from the results, the 2SS-GAM + cells scaffold had the best wound repair effect (see b, c in FIG. 9). This group had formed substantially intact epithelium at 15 days compared to the other groups, and had new hair follicle generation (see d in fig. 9). The 2SS-GAM + cells group had the largest blue area percentage in Masson trichrome staining (see e in FIG. 9, FIG. 10), demonstrating the highest area percentage of collagen fibers in this group.

The results show that the biological 3D printed 2SS-GAM inorganic-organic composite living cell scaffold has an outstanding effect in the process of repairing the diabetic wound surface, and has great application value and wide application prospect in the aspect of treating chronic difficult-to-heal skin wound.

Claims

1. An inorganic-organic composite living cell scaffold, which is characterized in that the living cell scaffold comprises a scaffold integral framework formed by constructing inorganic-organic composite biological ink and a plurality of cells which are distributed in a layered manner in the three-dimensional space of the scaffold integral framework; the upper half part of the living cell scaffold is in a layer-by-layer alternate arrangement mode of (A-C) y, A is inorganic-organic composite biological ink, and C is upper cells of the scaffold; the lower half part is in a layer-by-layer alternate arrangement mode of (A-B) x, wherein A is inorganic-organic composite biological ink, and B is cells on the lower layer of the bracket; x and y represent cycle periods which are alternately arranged and are positive integers;

the upper layer cells of the scaffold are fibroblasts, and the lower layer cells of the scaffold are vascular endothelial cells;

the inorganic-organic composite bio-ink comprises a bioactive inorganic material and a hydrogel matrix with strength and toughness; the bioactive inorganic material is hexagonal prism SrSiO which releases trace elements Si and Sr necessary for human body in physiological environment₃Inorganic fine particles; the mass ratio of the bioactive inorganic material to the hydrogel matrix is 0.7-4%;

spraying the cells on the inorganic-organic composite bio-ink through a miniature piezoelectric sample application needle.

2. The scaffold according to claim 1, wherein x: y = 3: 7-7: 3.

3. the live cell scaffold according to claim 1, wherein the hydrogel matrix is one of gellan gum, hyaluronic acid, sodium alginate, methyl cellulose, collagen; or the hydrogel matrix is a uniform mixture formed by physically combining multiple of gellan gum, hyaluronic acid, sodium alginate, methyl cellulose and collagen.

4. A method for preparing a scaffold for living cells according to any one of claims 1 to 3, comprising the steps of:

preparing inorganic-organic composite biological ink;

preparing the lower half part of a living cell scaffold which is alternately arranged layer by layer into (A-B) x by using inorganic-organic composite biological ink and cell suspension 1 by using an extrusion biological 3D printing method; similarly, the upper half part of the living cell scaffold which is alternately arranged layer by layer is (A-C) y by using the inorganic-organic composite biological ink and the cell suspension 2 and adopting an extrusion biological 3D printing method;

and (4) crosslinking and curing the printed scaffold to obtain the inorganic-organic composite living cell scaffold.

5. Use of the living cell scaffold of any one of claims 1 to 3 for culturing a skin tissue engineering construct in vitro.