CN115990291A - Biological ink, cell scaffold capable of immunoregulation and hair follicle regeneration promotion, and preparation method and application thereof - Google Patents
Biological ink, cell scaffold capable of immunoregulation and hair follicle regeneration promotion, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a biological ink, a cell scaffold capable of immunoregulating hair follicle regeneration and a preparation method and application thereof. The biological ink comprises hydrogel solution and living cells, and the concentration of the cells in the biological ink is (1-10) multiplied by 10 6 individual/mL; the hydrogel solution comprises an organic high polymer material serving as a hydrogel matrix and an inorganic component calcium molybdate for releasing functional ions; in the hydrogel solution, the mass fraction of the hydrogel matrix is 2-12% (w/v), and the mass concentration of calcium molybdate is below 1000 mug/mL. The cell scaffold simulates the microenvironment of cells in the healing of skin defects in vitro, rebuilds the hair follicle accessory structure in an animal skin defect model, and further promotes complete skin regeneration.
Description
Technical Field
The invention relates to a biological ink, a cell scaffold capable of immunoregulation and hair follicle regeneration, a preparation method and application thereof, and belongs to the technical field of biology.
Background
The skin is used as the first line of defense of the human body, and severely damaged skin cannot be completely regenerated. The lack of structures of skin appendages such as hair follicles can affect the functions of regulating body temperature, secretion and the like of skin. Follicular regeneration is critical to achieving regeneration of the functional accessory structures of the skin. Autograft is the best therapeutic approach to treat wound healing and follicular regeneration, however, the sources of autologous donors are limited. There is therefore an urgent need to develop a novel skin substitute having a hair follicle regenerating function.
Disclosure of Invention
The invention aims to provide a biological ink, a cell bracket capable of immunoregulation and hair follicle regeneration, a preparation method and application thereof, which simulate the microenvironment of cells in the healing of skin defects in vitro so as to promote the regeneration of the skin, in particular to the reconstruction of hair follicles with accessory structures.
In a first aspect, the present invention provides a bio-ink. The biological ink comprises hydrogel solution and living cells, and the concentration of the cells in the biological ink is (1-10) multiplied by 10 6 individual/mL; the hydrogel solution comprises an organic high polymer material serving as a hydrogel matrix and an inorganic component calcium molybdate for releasing functional ions; in the hydrogel solution, the mass fraction of the hydrogel matrix is 2-12% (w/v), and the mass concentration of calcium molybdate is below 1000 mug/mL.
Preferably, the cells include at least one of hair follicle stem cells, dermal papilla cells, endothelial cells, macrophages, neutrophils.
Preferably, the organic polymer material used as the hydrogel matrix comprises at least one of gellan gum, hyaluronic acid, methylcellulose, gelatin, methacrylic acid acylated hyaluronic acid, chitosan and sodium alginate; preferably, the hydrogel is a composite hydrogel with gelatin and methacrylic acid acylated gelatin as a matrix.
Preferably, the calcium molybdate has a diameter of 100 to 300nm, preferably 200nm.
Preferably, the hydrogel solution further comprises a photocrosslinking agent; the photo-crosslinking agent accounts for 0.1-0.4% (w/v) of the mass fraction of the hydrogel solution; more preferably, the photocrosslinker comprises at least one of Irgacure 2959, phenyl-2, 4, 6-trimethylbenzoyl lithium phosphonate (LAP).
In a second aspect, the present invention provides a cell scaffold that can immunomodulate follicle-stimulating regeneration. The cell scaffold capable of immunoregulating and promoting hair follicle regeneration is a three-dimensional scaffold frame prepared by 3D printing the biological ink according to any one of the above; the three-dimensional stent frame has a multi-layer structure printed by biological ink.
Preferably, the bio-ink forming the layer structure is a bio-ink having the same kind of cells.
Preferably, the bio-ink forming the layer structure is a bio-ink having different kinds of cells; preferably, the three-dimensional support frame has ase:Sub>A layer structure formed by stacking same-layer units-A-B-printed by two biological inks in ase:Sub>A mode of-A-B-A-B-circulation arrangement; more preferably, a is a bioeink containing hair follicle stem cells and B is a bioeink containing macrophages. The scaffold is characterized by comprising stem cells with tissue differentiation potential and macrophages for providing an immune microenvironment. After subsequent culture or implantation in vivo, the scaffold gradually releases molybdate ions, indirectly regulates stem cells by directly stimulating the stem cells and stimulating immune cells, and promotes skin and hair follicle regeneration.
In a third aspect, the present invention provides a method of preparing a cell scaffold that can immunomodulate follicle-stimulating regeneration. The biological ink is deposited by 3D printing in a staggered manner to form a layer structure of the scaffold, and then the scaffold is promoted to be crosslinked, especially to be photocrosslinked, so that the cell scaffold capable of regulating immunity and promoting hair follicle regeneration is obtained.
In a fourth aspect, the present invention provides the use of an immunoregulatory follicle-stimulating regenerative cell scaffold according to any of the preceding claims in the field of promoting skin repair and regeneration of skin tissue, in particular in the field of regeneration of hair follicles of the skin appendages.
Drawings
Figure 1 characterizes the calcium molybdate nanoparticles and slurries of the present invention. (a) An X-ray diffraction analysis chart obtained by an X-ray diffractometer (D/max 2550V, rigaku, japan); (b) Scanning electron micrographs, obtained by scanning electron microscopy (Magellan 400, FEI, usa); (c) The Ca, O and Mo element distribution diagram of the calcium molybdate nano-particles is obtained by a transmission electron microscope (JEM-2100F, japan) and a matched energy spectrometer (EDS); (d) Viscosity-shear rate curves for slurries containing varying concentrations of calcium molybdate; the peak value of each curve is 1000 mug/mL, 100 mug/mL, 500 mug/mL, 50 mug/mL, 250 mug/mL and 0 mug/mL from top to bottom in sequence; (e) Printing macroscopic photographs of the bracket by using the biological ink containing calcium molybdate with different concentrations; (f) Mo ion release curve (0 CM-GG, 50CM-GG, 250CM-GG, 500CM-GG from bottom to top) during stent culture.
FIG. 2 is a representation of the cellular activity of the biological 3D printed single culture scaffolds for hair follicle stem cells of the present invention. (a) Cell proliferation activity of hair follicle stem cells after 1, 7, 14 and 21 days of single culture bracket culture; in the bar graph, 0 μg/mL, 50 μg/mL, 100 μg/mL, 250 μg/mL, 500 μg/mL, 1000 μg/mL are sequentially from left to right. (b) Cell live and dead staining images after 1, 7, 14 and 21 days of culture of the hair follicle stem cells by the single culture bracket are marked with a scale of 500 mu m.
FIG. 3 is a representation of the cellular activity of the biological 3D printed hair follicle stem cell single culture scaffold of the present invention. (a) The hair follicle stem cells are subjected to nuclear-skeleton staining after 1, 7, 14 and 21 days of culture by the single culture bracket of the hair follicle stem cells, and the scale is 200 mu m; (b) The gene expression condition of the hair follicle stem cells is detected by qRT-PCR after the hair follicle stem cells are cultured for 7 days by a single culture bracket, and 0CM-GG-D, 50CM-GG-D, 250CM-GG-D and 500CM-GG-D are sequentially arranged from left to right in the histogram.
FIG. 4 is a representation of the cellular activity of the biological 3D printed macrophage single culture scaffold of the invention. (a) Cell proliferation activity after 1, 7, 14, 21 days of macrophage single culture rack culture; in the bar graph, 0 mug/mL, 50 mug/mL, 100 mug/mL, 250 mug/mL, 500 mug/mL, 1000 mug/mL are sequentially arranged from left to right; (b) Cell live/dead staining images after 1, 7, 14, 21 days of macrophage single culture scaffold culture were on a scale of 500 μm.
FIG. 5 is a representation of the cellular activity of the biological 3D printed macrophage single culture scaffold of the invention. (a) Immunofluorescence images of macrophage M1 phenotype marker (CCR 7) and M2 phenotype marker (CD 206) after 7 days of macrophage single culture scaffold culture; (b) Representative images of immunofluorescence of pure macrophage CD206 isolated from macrophage single culture scaffold.
FIG. 6 is a representation of the cellular activity of the biological 3D printed macrophage single culture scaffold of the invention. (a) statistics of CD206 corresponding fluorescence intensity expression; (b) The expression of CCR7 and CD206 genes obtained by qRT-PCR detection after 7 days of macrophage single culture rack culture is 0CM-GG-R, 50CM-GG-R, 250CM-GG-R and 500CM-GG-R in sequence from left to right in the histogram.
FIG. 7 is a representation of a biological 3D printed co-cultured scaffold containing hair follicle stem cells and macrophages in accordance with the present invention. (a) co-cultivation scaffold printing schematic; (b) Top view of cell fluorescence images of co-culture scaffolds on day 1 and day 7; (c) The single culture and co-culture bracket culture of hair follicle stem cells show the expression condition of hair follicle growth markers (PDGF-alpha, PDGF-beta, C-Myc) and angiogenesis growth factor (VEGF) genes after 7D, and the single culture and co-culture bracket culture are Mono-D, co-D-1/5R, co-D-1/3R, co-D-1/2R in sequence from left to right in the histogram; (d) The Co-culture scaffolds were cultured for 7 days for Igf-1 and Hgf gene expression in macrophages, and Co-D-1/5R, co-D-1/3R, co-D-1/2R was sequenced from left to right in the histogram.
FIG. 8 is a graph depicting the results of skin repair of a multicellular inorganic-organic material composite bioactive scaffold of the present invention in a nude mouse skin defect. (a) immunofluorescent staining of iNOS at day 7, scale 50 μm; (b) Immunofluorescence staining of Arg-1 on day 7, scale 50 μm; (c) day 30H & E staining with a scale of 200 μm; (d) a macroscopic photograph of the wound on day 40, scale 1cm; (e) immunofluorescent staining fluorescence intensity statistics of iNOS; (f) immunofluorescent staining fluorescence intensity statistics of Arg-1; (g) day 30 skin thickness statistics; (h) statistics of number of new hair follicles on day 30. (e) In the bar graphs of (f), (g) and (h), blank, GG, GG-D, CM-GG-D and CM-GG-Co are sequentially arranged from left to right. Wherein Blank is a stentless set, GG is a pure stent set, GG-D is a stent set containing hair follicle stem cells, CM-GG-D is a stent set containing calcium molybdate and hair follicle stem cells, and CM-GG-Co is a stent set containing calcium molybdate, hair follicle stem cells and macrophages.
FIG. 9 is a graph depicting the results of skin reconstruction and hair regeneration of a multi-cell containing inorganic-organic material composite bioactive scaffold for a black rat skin defect. (a) Macroscopic images of the surgical site at specific time points within 40 days of 0, 10, 25 and 40 days, with a scale of 5mm; (b) day 0 and day 40 black mouse panoramic images; (c) neonatal hair coverage statistics.
FIG. 10 is a graph depicting immunofluorescent staining results of a multi-cell containing inorganic-organic material composite bioactive scaffold for skin defects in black rats. (a) Ki67 immunofluorescence staining of day 20 sections, scale 200 μm; (b) H & E immunofluorescent staining of day 20 sections, scale 200 μm; (c) day 40 neonatal power generation mirror plot, scale is 10 μm; (d) Ki67 positive cell statistics; (e) statistics of number of new hair follicles on day 20; (f) statistics of diameter of new hair on day 40. (d) In the bar graph of (e) and (f), blank, GG, GG-Co, CM-GG, CM-GG-Co are sequentially arranged from left to right. Wherein Blank is a stentless set, GG is a pure stent set, GG-D is a stent set containing hair follicle stem cells, CM-GG-D is a stent set containing calcium molybdate and hair follicle stem cells, and CM-GG-Co is a stent set containing calcium molybdate, hair follicle stem cells and macrophages.
Detailed Description
The invention is further illustrated by the following embodiments, which are to be understood as merely illustrative of the invention and not limiting thereof. Unless otherwise specified, each percentage refers to a mass percent.
The bio-ink comprises a hydrogel solution and living cells, wherein the hydrogel solution comprises an organic high polymer material serving as a hydrogel matrix and an inorganic component calcium molybdate for releasing functional ions.
The organic polymer material used as the hydrogel matrix has good biocompatibility and should satisfy the following requirements: 1) The liquid crystal display device has temperature-sensitive property of changing physical state along with temperature change, can be dissolved in water at high temperature, and the obtained aqueous solution can flow in a cell-friendly temperature range to enable living cells to be uniformly mixed into the liquid crystal display device, and can be in gel state to facilitate space position fixation of the cells and subsequent extrusion printing. 2) The material has good shear thinning and forming performance, namely the viscosity of the material is reduced when the air pressure applied to the material is increased, so that the material is smoothly extruded from a printing material cylinder to form a three-dimensional structure designed by computer modeling software. 3) The good material exchange capacity ensures that nutrient components in the culture medium can be transmitted to cell parts in the long-time culture process, and meanwhile, metabolic products of cells can be discharged out of the bracket in time.
The organic polymer material with good biocompatibility comprises at least one of gellan gum, hyaluronic acid, methylcellulose, gelatin, methacrylic acid acylated hyaluronic acid, chitosan and sodium alginate. When in use, one or more organic polymer materials meeting the characteristics are mixed with water or PBS according to the proportion to form hydrogel solution capable of wrapping cells. The solution has the shear thinning property for extrusion printing and the external stimulus response crosslinking property.
Preferably, the hydrogel is a composite hydrogel with gelatin and methacrylic acid acylated gelatin as a matrix. The composite hydrogel can provide crosslinking action excited by a photoinitiator after extrusion printing to enable the stent to maintain a specific shape, and can enhance the substance transmission performance of the stent through dissolution of gelatin in long-term culture. In some embodiments, gelatin, methacrylic acid acylated gelatin are mixed to form a hydrogel matrix for the bio-ink. In some embodiments, the hydrogel matrix comprises 2-12% (w/v) of the hydrogel solution by mass. In order to achieve both cell compatibility and printability of the hydrogel, the mass fraction of hydrogel matrix in the hydrogel solution is preferably 4-8% (w/v). When in use, the organic polymer material can be dissolved in deionized water or PBS to form a solution.
Gelatin is heated and dissolved, can absorb 5-10 times of water to expand and soften, and becomes gel after cooling. The cell culture medium can be gradually dissolved and separated from the bracket under the condition of cell culture, so that the bracket has more pores for providing oxygen and nutrition exchange. As an example, gelatin may be present in the hydrogel solution at a concentration of 1-6% (w/v). The methacrylic acid acylated gelatin is modified by gelatin, so that the thermosensitive property of the gelatin is reserved, meanwhile, the methacrylic acid acylated gelatin can be matched with a photoinitiator, is crosslinked in a friendly way under the illumination of a specific wavelength range, and is used as a support in the culture process of a bracket, so that a three-dimensional structure with certain strength is formed. As an example, the concentration of the methacrylic acid acylated gelatin is 1-6% (w/v) of the hydrogel solution.
The mass concentration of calcium molybdate in the hydrogel solution is 1000 mug/mL or less, preferably 50-500 mug/mL. Too high a calcium molybdate concentration can have side effects that impair cell viability in the scaffold. The diameter of the inorganic material calcium molybdate may be 100-300nm. Calcium molybdate nanoparticles having a diameter of 200nm are preferred.
Calcium molybdate may be synthesized by a microemulsion process. Adding the Mo source solution and the Ca source solution into the organic mixed solution respectively, stirring to be transparent to obtain two equal-amount microemulsions, slowly and uniformly dripping the Ca source-containing microemulsion solution into the Mo source microemulsion solution, aging the obtained mixture at room temperature for 12-36 hours, respectively washing the mixture with absolute ethyl alcohol and deionized water for three times, and freeze-drying the mixture for 12-36 hours at the sintering temperature of 300-900 ℃ for 4-8 hours. The organic mixed solution is at least 3 of cyclohexane, cetyltrimethylammonium bromide, triton-100, 1-pentanol and 1-octanol, preferably a mixed solution of cyclohexane (200-300 mL), cetyltrimethylammonium bromide (5-20 g) and 1-pentanol (5-30 mL). The Mo source is molybdate, and the Ca source is CaCl 2 、Ca(NO 3 ) 2 At least one of them. Preferably, the Mo source is 0.05-0.15mol/LNa 2 MoO 4 ·2H 2 The concentration of O and Ca source is 0.05-0.15mol/L CaCl 2 Aqueous solutions, respectively dissolved in the organic mixed solution, form two types of microemulsion solutions.
In some embodiments, the hydrogel solution further comprises a photocrosslinking agent. The photocrosslinking agent includes, but is not limited to, at least one of Irgacure 2959, phenyl-2, 4, 6-trimethylbenzoyl lithium phosphonate (LAP). LAP is preferred because it has higher water solubility, is excitable by blue light at 405nm, and has better biocompatibility. As an example, LAP accounts for 0.1-0.4% (w/v) of the mass fraction of the hydrogel solution. Preferably, LAP is present in the hydrogel solution at a mass fraction of 0.25% (w/v) to minimize the toxicity and crosslinking of the initiator to the cells.
The cells include, but are not limited to, at least one of hair follicle stem cells, dermal papilla cells, endothelial cells, macrophages, neutrophils. The cells may be used as a viable cell suspension. The concentration of cells in the biological ink is (1-10) x 10 6 personal/mL, excellentSelected as (1.2-6) x 10 6 And each mL.
The preparation method of the bio-ink is also described.
A hydrogel solution (also referred to as a slurry) is prepared. The calcium molybdate nano-particles are sterilized by ultraviolet rays for 2 to 6 hours in advance. 2% -4% (w/v) of methacrylic acid acylated gelatin, 2% -4% (w/v) of gelatin and 0.1% -0.4% of LAP (w/v) are dissolved in water or PBS, and the mixture is heated in a water bath at 50-70 ℃ for 20-40 minutes until the mixture is completely dissolved. The hot solution was rapidly filtered using a filter membrane and sterile syringe, then calcium molybdate nanoparticles were added to the solution (0-1000 ug/mL concentration, respectively), and finally the slurry was stored in a sterile 4 ℃ environment in a sealed, light-tight manner.
Preparing the biological ink. The slurry was warmed to 37 ℃ for use. The cells were then dispersed in the slurry for later use. Taking hair follicle stem cells as an example, digesting, centrifuging, collecting and dispersing hair follicle stem cells in slurry to obtain a cell concentration of (1.2-6) ×10 6 The suspension of each mL is used for obtaining the biological ink A. Taking macrophage as an example, centrifuging and collecting macrophage and dispersing in slurry to obtain a cell concentration of (1.2-6) ×10 6 And (3) obtaining the biological ink B from the suspension of each mL. The bio-ink was transferred to a metal cartridge and cooled at 4 ℃ for 10-30 minutes. Refrigeration can form gel states that can be used for extrusion printing.
The cell scaffold (also called as inorganic-organic material composite bioactive scaffold containing multicellular) capable of immunoregulating and promoting hair follicle regeneration has the three-dimensional scaffold frame printed by the bioeink in a 3D mode. As an example, the three-dimensional stent frame is layered inbase:Sub>A single bio-ink according to-base:Sub>A- (or-B-) or two bio-inks according to-base:Sub>A-B-cyclic arrangement pattern. A is biological ink containing hair follicle stem cells, and B is biological ink containing macrophages. The scaffold can effectively promote cell proliferation and differentiation activity, and simultaneously provides an immune microenvironment for promoting hair follicle regeneration. Through testing, cells can be continuously proliferated and spread in the culture process for three weeks, macrophages are polarized to an M2 phenotype, and hair follicle stem cells express cytokines related to the growth period of hair follicles.
The immunoregulatory follicle-stimulating regenerative cell scaffold preferably has a layered structure of staggered layers, stacked layers. For example, the product (-A-B-) obtained by printing two kinds of ink containing different kinds of cells at one time is regarded as a unit of the same layer. The intersection of two bio-ink extruded filaments containing different types of cells in the same layer unit is 90 deg., for example, fig. 7a may be considered as a same layer unit. The different units of the same layer are different layers. The different layers are stacked in the same orientation in the spatial vertical direction, i.e. the projections of the different layers in the x-y plane are completely overlapping.
Therefore, the cell scaffold is an inorganic-organic material composite bioactive scaffold containing multiple cells, and the inorganic component (calcium molybdate nano particles) can promote hair follicle regeneration through immunoregulation of microenvironment, so that the cell scaffold has potential application value for treating severely damaged skin lacking accessory organs. The skin-like scaffold is formed by the mode of staggered layers and stacked layers in the same layer on the space structure, crosstalk between cells is realized through paracrine action while the cells are not in direct contact, and meanwhile, the immunoregulation action of the inorganic material calcium molybdate and the differentiation promoting action on stem cells synergistically induce regeneration of hair follicle structures.
The three-dimensional structure of the cell scaffold capable of immunoregulating and promoting hair follicle regeneration has the reduction degree, the fidelity and the stability within an acceptable range. The stability includes the ability to maintain shape for a short period of time after the ink is extruded, and the ability to maintain the shape of the scaffold during subsequent culturing. According to the invention, through the space design of materials and cells, molybdate ions are released to directly stimulate hair follicle stem cells, and also through stimulating immune cells to generate mediating factors, hair follicle stem cells are indirectly stimulated. The two modes act together to promote the expression of genes associated with differentiation of hair follicle stem cells into hair follicles.
Skin defects are accompanied by a massive loss of tissue cells. Effective wound healing, especially regeneration of accessory structures, cannot be achieved if the proliferation and nutrient supply of cells alone is by itself. The cell scaffold capable of immunoregulation and hair follicle regeneration provides a more careful in vitro skin-like structure scheme, which comprises cell-containing biological ink approaching to in vivo cell density, controllable scaffold thickness, adjustable cell proportion and proper addition amount of inorganic materials. Biological 3D printing provides conditions for in-vitro delivery of exogenous cells, hydrogel simulates activities such as proliferation, migration, differentiation and the like of extracellular matrix maintenance cells, and design of a printing model provides a customized effect for cell space arrangement. The scaffold wraps the cells, which is beneficial to directional delivery of the cells and long-term maintenance of the wound site. Skin injury thickness that different causes caused is different, and through the regulation control layer number and the thickness of printing parameter, accurate preparation required support avoids extravagant. In addition, the paracrine effect can be improved by adjusting the proportion of stem cells and immune cells and the doping concentration of the functional inorganic material, so that a better hair follicle regeneration effect can be achieved.
The method for preparing the cell scaffold for immunoregulation and hair follicle promotion according to the present invention will be described. The biological ink is deposited by 3D printing in a staggered manner to form a layer structure of the scaffold, and then the scaffold is promoted to be crosslinked, especially to be photocrosslinked, so that the cell scaffold capable of regulating immunity and promoting hair follicle regeneration is obtained. The layer structure may be formed by pneumatic extrusion through a 3D printer.
For single culture cell scaffolds: transferring the cooled biological ink A to a printer system, and setting parameters: the temperature of the charging barrel is controlled to be 15-25 ℃; the outer diameter of the bracket is 5-15mm; the number of layers is 2-10; the pressure is 20-50kPa. Under the action of pneumatic extrusion, the biological ink A depositsbase:Sub>A first layer of hydrogel fine prism alongbase:Sub>A freezing platform (the temperature is 0-10 ℃), then rotates 90 degrees to depositbase:Sub>A second layer, and then rotates 90 degrees anticlockwise to depositbase:Sub>A third layer … …, namely, the A cell single culture scaffold is prepared by printing the A cell single culture scaffold inbase:Sub>A 90-degree staggered mode. After printing, the stent is irradiated with 405nm blue light for 30-90s, so that the stent shape is fixed. And then transferred to an incubator for subsequent culturing. In the same manner, a B cell single culture scaffold can be prepared.
For co-cultured cell scaffolds: transferring the cooled biological ink A and B to channels at different positions of a printer system, and setting parameters: the temperature of the charging barrel is controlled to be 15-25 ℃; the outer diameter of the bracket is 5-15mm; the number of layers is 2-10; the pressure is 20-50kPa. The biological ink A is deposited with hydrogel fine prisms of A along ase:Sub>A freezing platform (the temperature is 0-10 ℃) under the action of pneumatic extrusion, then the biological ink B is deposited with hydrogel fine prisms of A in ase:Sub>A 90-degree crossed mode under the action of pneumatic extrusion, and then the biological ink A, B is circularly deposited … …, namely, two cell co-culture scaffolds are prepared by printing in an arrangement mode of-A-B-A-B-. After printing, the stent is irradiated with 405nm blue light for 30-90s, so that the stent shape is fixed.
The invention also provides application of the cell scaffold capable of immunoregulating and promoting hair follicle regeneration in the fields of promoting skin repair and skin tissue regeneration, in particular application in the field of skin accessory structure-hair follicle regeneration.
The present invention will be described in more detail by way of examples. It is also to be understood that the following examples are given solely for the purpose of illustration and are not to be construed as limitations upon the scope of the invention, since numerous insubstantial modifications and variations will now occur to those skilled in the art in light of the foregoing disclosure. The specific process parameters and the like described below are also merely examples of suitable ranges, i.e., one skilled in the art can make a suitable selection from the description herein and are not intended to be limited to the specific values described below.
Example 1
Preparation of calcium molybdate nanoparticles
2 parts of the same organic mixed solution (cyclohexane 250mL, cetyltrimethylammonium bromide 10g, n-amyl alcohol 10 mL) was prepared. Respectively adding 0.1mol/L Na 2 MoO 4 ·2H 2 O and 0.1mol/L CaCl 2 The aqueous solution was dissolved in the above solution and stirred to be transparent to form two microemulsion solutions C and D. The microemulsion solution D was continuously stirred on a magnetic stirrer. The microemulsion solution C was slowly and uniformly dropped into the microemulsion solution D through a separating funnel. The mixed solution was aged at room temperature for 24 hours, centrifuged, washed 3 times with absolute ethanol and deionized water, respectively, and the white product was freeze-dried for 24 hours. Finally calcining for 6 hours in an electric furnace at 600 ℃, removing organic impurities, and sealing and preserving for standby.
Fig. 1 (a), (b) and (c) are characterization results of calcium molybdate nanoparticles obtained according to the above preparation method. The X-ray diffraction pattern shows thatIts phase is pure CaMoO 4 Corresponds to PDF #85-1267 (a). The scanning electron microscope image showed that calcium molybdate was approximately spherical (b) with a diameter of about 200nm. Elemental scanning images observed a uniform distribution of Ca, mo and O elements on the calcium molybdate surface, confirming the successful preparation of calcium molybdate nanoparticles (c).
Example 2
Preparation of biological ink
The Calcium Molybdate (CM) nano-particles are sterilized by ultraviolet irradiation for 4 hours in advance and then are placed in a sterile super clean bench for standby. 3% of methacrylic acid-acylated gelatin (w/v), 3% gelatin (w/v) and 0.25% LAP (w/v) were dissolved in PBS solution, placed in a water bath at 60℃until complete dissolution, during which the solutions were mixed uniformly (GG) by means of a shaker. The hot solution was filtered rapidly with a filter and 20mL sterile syringe, and then the calcium molybdate nanoparticles were added to the solution and mixed thoroughly with a pipette (calcium molybdate addition concentrations of 0, 50, 100, 250, 500, 1000 μg/mL). Finally, the bio-ink is sealed and stored at 4 ℃ in a dark place for later use.
Fig. 1 (d), (e) and (f) show the results of the bio-ink prepared by the above preparation method. Rheological testing showed that all bioinks have shear thinning properties. Furthermore, the more calcium molybdate nanoparticles are added, the higher the viscosity of the bio-ink (d) under the same shear rate conditions. From the photograph (e) of the stent containing various concentrations of calcium molybdate nanoparticles, an increase in calcium molybdate concentration was observed, the stent shape was more regular, and the printing performance was remarkably improved. The ion release result shows that the calcium molybdate-containing stent can slowly release molybdate ions, and the release amount and the doping amount are positively correlated (f).
Example 3
Biological 3D prints cell list and cultivates support
The prepared bio-ink was heated to 37 ℃ and mixed with cells to obtain a uniform cell-loaded bio-ink, using hair follicle stem cells (6×10, respectively 6 individual/mL) and macrophages (3X 10) 6 and/mL). The cell-loaded bio-ink was then transferred to a sterile pneumatic squeeze metal syringe equipped with a 27G needle (inner diameter: 210 μm) and a temperature control system. Refrigerating at 4deg.CAfter 20 minutes, the printer system temperature was set to 20℃and the pressure was set to 30kPa, and printing of the cell-loaded scaffolds was started. The bio-ink was extruded onto a 4 ℃ sterile slide and immediately crosslinked for 30 seconds by irradiation with 405nm blue light. The bioprinted scaffolds were placed in culture medium of the corresponding cells for subsequent culture. The new media was replaced about 2 hours after bioprinting and further replaced before the media nutrients were consumed.
FIGS. 2-3 characterize cell activity in a single culture scaffold of hair follicle stem cells. CCK-8 results (FIG. 2 a) demonstrate that cell proliferation is down-regulated in single culture scaffolds containing 100-1000. Mu.g/mL, especially in 500 and 1000. Mu.g/mL calcium molybdate, while cell proliferation is enhanced in the 50. Mu.g/mL group compared to the 0. Mu.g/mL group. The cell activity of the 100-500 μg/mL group was maintained at more than 90% of that of the 0 μg/mL group by day 21 of culture. Cell viability/death staining assays (FIG. 2 b) showed that each group of cells was dotted on day one, then spread out and began to grow on the stent surface on day 7, especially 250CM-GG-D and 500CM-GG-D groups. On day 14, cells proliferated so rapidly at a location suitable for aggregation (e.g., at the junction of different prisms) that they dropped from the scaffold after 21 days of culture due to excessive density. The morphology of the cells in the single culture scaffolds of hair follicle stem cells was observed by actin immunofluorescence staining (fig. 3 a), the scaffold was clearly stained after 7 days, and these cells grew well and spread continuously with the extension of the culture days. The PCR results showed relative gene expression levels (FIG. 3 b), with 50CM-GG-D, 250CM-GG-D and 500CM-GG-D all significantly upregulating mRNA expression of PDGF- α, C-Myc and VEGF compared to the 0CM-GG-D group. Although there was no significant difference in PDGF-beta expression from 0CM-GG-D in 50CM-GG-D, PDGF-beta expression was significantly enhanced in 250CM-GG-D and 500CM-GG-D.
FIG. 4 shows cell activity in a macrophage single culture scaffold. After 21 days of culture, single culture scaffolds containing 50 and 250. Mu.g/mL calcium molybdate significantly promoted macrophage proliferation (FIG. 4 a). Live/dead staining (fig. 4 b) showed that calcium molybdate also had good cell compatibility with macrophages. Immunofluorescent staining showed that groups 0CM-GG-D, 50CM-GG-D and 250CM-GG-D expressed more CD206, indicating the presence of more M2-type macrophages (FIG. 5 a). To further demonstrate that macrophages in scaffolds were isolated, collected and subjected to CD206 immunofluorescent staining (FIG. 5), semi-quantitative analysis (FIG. 6 a) showed that the fluorescence intensities of CD206 were significantly up-regulated for 50CM-GG-D, 250CM-GG-D and 500CM-GG-D compared to 0CM-GG-D, especially for the 250CM-GG-D group CD206 expression was strongest. Furthermore, the PCR gene expression results were consistent with the immunofluorescent staining results (fig. 6 b). Overall, these results demonstrate that calcium molybdate has the ability to stimulate CD206 production while inhibiting CCR7 production, demonstrating the effectiveness of calcium molybdate in stimulating macrophage transition towards the M2 phenotype.
Example 4
Biological 3D prints cell co-culture support
A slurry was prepared at a calcium molybdate concentration of 250. Mu.g/mL, heated to 37℃and mixed with cells to obtain a uniform cell-loaded bio-ink, using hair follicle stem cells (6X 10, respectively 6 individual/mL) and macrophages (0 x 10) 6 Per mL, 1.2X10) 6 Per mL, 2X 10 6 3X 10 of the total volume of the solution per mL 6 and/mL). The cell-loaded bio-ink was then transferred to a sterile pneumatic squeeze metal syringe equipped with a 27G needle (inner diameter: 210 μm) and a temperature control system. After cold storage at 4℃for 20 minutes, the printer system temperature was set to 20℃and the pressure was set to 30kPa, and printing of the cell-loaded scaffolds was started. The method comprises the steps of depositing ase:Sub>A first layer of hydrogel fine prisms along ase:Sub>A freezing platform (the temperature is 4 ℃) under the action of pneumatic extrusion, then depositing ase:Sub>A second layer of hydrogel fine prisms at 90 DEG with the first layer under the action of pneumatic extrusion by macrophage biological ink, and then printing the two biological inks A, B in ase:Sub>A circulating way … …, namely in ase:Sub>A 90 DEG staggered way of-A-B-A-B-, to prepare the cell Co-culture bracket (Co-D-1/5R, co-D-1/3R, co-D-1/2R). When the macrophage concentration was 0X 10 6 Single culture scaffolds (Mono-D) were used per mL. After printing, the stent was irradiated with 405nm blue light for 30-90s, so that the stent shape was fixed, the bio-ink was extruded onto a 4 ℃ sterile slide, and immediately crosslinked for 30 seconds by 405nm blue light irradiation. The bioprinted scaffolds were placed in culture medium of the corresponding cells for subsequent culture. The new media was replaced about 2 hours after bioprinting and further replaced before the media nutrients were consumed.
FIG. 7 shows that the co-cultured scaffolds printed were designed to meet the expectations. The two cells were alternately arranged at 90℃and the arrangement was then maintained stably until the seventh day of culture (FIGS. 7a, b). Compared to single culture scaffolds with hair follicle stem cells only (Mono-D), co-culture scaffolds with macrophages (Co-D-1/5R, co-D-1/3R, co-D-1/2R) secreted more of the hair follicle growth phase-associated cytokines (FIG. 7 c), with optimal effect when the number of macrophages is 1/3 of the number of hair follicle stem cells; the expression of macrophage Igf-1 and Hgf (FIG. 7 d) is also consistent with the above results.
Example 5
Multi-cell inorganic-organic material composite bioactive stent implanted into skin defect of nude mice
SPF-grade male BALB/c-nude mice were used 6 weeks old. These nude mice were randomly divided into five groups: a no scaffold group (Blank group), a pure scaffold group (GG), a scaffold group containing hair follicle stem cells (GG-D), a scaffold group containing calcium molybdate and hair follicle stem cells (CM-GG-D), a scaffold group containing calcium molybdate, hair follicle stem cells and macrophages (CM-GG-Co). All stents used for implantation were 10mm in diameter and 1mm in thickness. The nude mice were first anesthetized, the skin on the back thereof was sterilized with iodophor to obtain a circular wound with a diameter of 10mm, and then the stents were implanted into the wound site and fixed with sterile gauze and medical dressing. Nude mice were sacrificed 7 and 21 days after surgery, wound skin samples were collected for characterization by sectioning and immunofluorescent staining, H & E staining, and the like.
FIG. 8 shows the staining results of a multicellular inorganic-organic material composite bioactive scaffold for skin defect sections of nude mice. From the immunofluorescence staining (FIGS. 8a, b) and fluorescence intensity statistics (FIGS. 8e, f) of iNOS and Arg-1 at day 7, calcium molybdate-containing scaffolds (CM-GG-D and CM-GG-Co groups) had higher Arg-1 expression levels and lower iNOS expression levels. Corresponding to the results of in vitro experiments, scaffolds containing calcium molybdate induced polarization of macrophages towards the M2 phenotype and established an anti-inflammatory microenvironment around the wound. H & E staining on day 21 (FIG. 8 c) and corresponding skin thickness (FIG. 8 g) and number of new hair follicles (FIG. 8H) statistics indicate that all calcium molybdate-containing groups (CM-GG-D and CM-GG-Co groups) significantly enhanced hair follicle formation, while the blank and pure scaffolds did not observe hair follicle formation, with few new hair follicles in the scaffolds containing hair follicle stem cells. All groups containing hair follicle stem cells (GG-D, CM-GG-D and CM-GG-Co groups) have remarkably improved thickness of the newly generated full-thickness skin, and the adhesion between epidermis and dermis structures is tighter, so that the hair follicle stem cells have more complete skin structure. Significant hair production was observed in the CM-GG-D and CM-GG-Co groups on day 40, and no new hair was observed in the other groups (FIG. 8D).
Example 6
Multi-cell inorganic-organic material composite bioactive stent implanted into black rat skin defect
Male C57BL/6 black mice at 6 weeks of age. These black mice were randomly divided into five groups: a no scaffold group (Blank group), a pure scaffold group (GG), a scaffold group containing hair follicle stem cells and macrophages (GG-Co), a scaffold group containing calcium molybdate (CM-GG), a scaffold group containing calcium molybdate, hair follicle stem cells and macrophages (CM-GG-Co). First, hair on the back of a black mouse was removed using electric hair clipper, androgenic alopecia was induced using testosterone propionate, 5mg/mL testosterone propionate solution was prepared and injected on its back per day at 0.2 mL. After one week, a round wound with a diameter of 10mm was obtained on the back of the black mouse, and then the stent was implanted into the wound site and fixed with sterile gauze and medical dressing. Wound beds were observed 10, 25, 40 days after surgery and photographed to record hair regrowth, and hair regrowth area and coverage were measured by Image J procedure. After 20 days of operation, the black mice are sacrificed, wound skin samples are collected for characterization such as H & E, immunofluorescent staining and the like, and the hair growth condition is observed by a scanning electron microscope on the 40 th day.
FIG. 9 shows the results of skin reconstruction and hair regeneration using a multi-cell-containing inorganic-organic material composite bioactive scaffold for skin defects of black rats. Macroscopic images of the surgical site and statistics of the coverage of the new hairs at specific time points (0, 10, 25, 40 days) within 40 days are shown in fig. 6 a-b, on 40 days, blank and GG groups had no new hairs, GG-Co and CM-GG groups had small amounts of new hairs, CM-GG-Co groups had large coverage of new hairs, and on 25 days, the new hairs were observed in the GG-Co, CM-GG and CM-GG-Co groups. The above results indicate that the addition of double cells or calcium molybdate is effective in promoting hair regeneration of wound skin, which is also an important sign of full-thickness repair of skin. The direct stimulation and indirect immunomodulation of calcium molybdate promote hair follicle formation, and the addition of cells provides sufficient "ammunition" for hair follicle remodeling, and the combined use of the two further accelerates hair follicle and hair regeneration.
FIG. 10 shows immunofluorescent staining results of a multi-cell containing inorganic-organic material composite bioactive scaffold for skin defects in black rats. Ki67 is a marker of cell proliferation, and fig. 10 a and d are Ki67 immunofluorescent staining and statistical results of the sections, respectively. The Blank and GG groups showed only a small amount of Ki67 expression at the newly generated epidermis, and the Ki67 fluorescence of the GG-Co group had a remarkable spherical shape, indicating that the Ki67 fluorescence of the CM-GG and CM-GG-Co groups was significantly enriched in the root of hair follicle at the early stage of hair follicle formation, indicating that the cells associated with the anagen phase proliferated in a large amount. In combination with H & E staining and statistics of the number of new hair follicles (FIGS. 10b and E), the CM-GG and CM-GG-Co groups had more new hair follicles, and the area below the dermis layer was also near mature follicles, with intact skin structure. According to the black mouse hair electron microscope pictures and diameter statistics (fig. 10c and f) on day 40, the newly generated hair scales of the Blank and GG groups are incomplete, the diameter is not more than 15 microns, the hair scales of the other 3 groups are complete, and the hair scales are similar to the appearance and the size of normal hair of a black mouse. In conclusion, the addition of the cells and the calcium molybdate respectively remarkably promotes the full-layer skin construction effect of the skin defect, and the simultaneous addition effect of the cells and the calcium molybdate is optimal.
Claims (10)
1. A bio-ink is characterized in that the bio-ink comprises a hydrogel solution and living cells, and the concentration of the cells in the bio-ink is (1-10) multiplied by 10 6 individual/mL; the hydrogel solution comprises an organic high polymer material serving as a hydrogel matrix and an inorganic component calcium molybdate for releasing functional ions; in the hydrogel solution, the mass fraction of the hydrogel matrix is 2-12% (w/v), and the mass concentration of calcium molybdate is below 1000 mug/mL.
2. The bio-ink of claim 1 wherein the cells comprise at least one of hair follicle stem cells, dermal papilla cells, endothelial cells, macrophages, neutrophils.
3. The bio-ink according to claim 1 or 2, wherein the organic polymer material as the hydrogel matrix comprises at least one of gellan gum, hyaluronic acid, methylcellulose, gelatin, methacrylic acid acylated hyaluronic acid, chitosan, sodium alginate; preferably, the hydrogel is a composite hydrogel with gelatin and methacrylic acid acylated gelatin as a matrix.
4. A bio-ink according to any of claims 1 to 3, wherein the calcium molybdate has a diameter of 100-300nm, preferably 200nm.
5. The bio-ink according to any one of claims 1 to 4, wherein the hydrogel solution further comprises a photo-crosslinking agent; the photo-crosslinking agent accounts for 0.1-0.4% (w/v) of the mass fraction of the hydrogel solution; more preferably, the photocrosslinker comprises at least one of Irgacure 2959, phenyl-2, 4, 6-trimethylbenzoyl lithium phosphonate (LAP).
6. A cell scaffold capable of immunoregulation of follicle-stimulating regeneration, characterized in that the cell scaffold capable of immunoregulation of follicle-stimulating regeneration is a three-dimensional scaffold frame prepared by 3D printing with the bio-ink according to any one of claims 1 to 5; the three-dimensional stent frame has a multi-layer structure printed by biological ink.
7. The immunoregulatory follicle-stimulating cell scaffold of claim 6, wherein the bio-ink forming the layer structure is a bio-ink having cells of the same species.
8. The immunoregulatory follicle-stimulating cell scaffold of claim 6, wherein the bio-ink forming the layer structure is a bio-ink having cells of different species; preferably, the three-dimensional support frame has ase:Sub>A layer structure formed by stacking same-layer units-A-B-printed by two biological inks in ase:Sub>A mode of-A-B-A-B-circulation arrangement; more preferably, a is a bioeink containing hair follicle stem cells and B is a bioeink containing macrophages.
9. The method for preparing an immunoregulatory follicle-promoting cell scaffold according to any one of claims 6 to 8, characterized in that the bio-ink is deposited by 3D printing in a staggered manner to form a layered structure of the scaffold, and then the scaffold is caused to crosslink, in particular photocrosslink, to obtain the immunoregulatory follicle-promoting cell scaffold.
10. Use of an immunoregulatory follicle-stimulating regenerative cell scaffold according to any of claims 6 to 8 in the field of promoting skin repair and regeneration of skin tissue, in particular in the field of regeneration of hair follicles of the skin appendages.
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