CN110575567A - scaffold composite material and preparation method and application thereof - Google Patents
scaffold composite material and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a scaffold composite material and a preparation method thereof, which comprises the steps of preparing single cell suspension of mesenchymal stem cells, then mixing hydrolyzed placenta extract, biological macromolecules, degradable macromolecules and a solvent, adjusting the pH value to 7.2-7.4, and preparing to obtain a first scaffold material premixed solution; and finally, mixing the single cell suspension of the mesenchymal stem cells and the first scaffold material premixed solution according to a preset formula to obtain the scaffold composite material. The invention also discloses application of the bracket composite material. The embodiment of the invention applies the technology of combining the mesenchymal stem cells and the traditional scaffold, and the prepared scaffold composite material has the advantages of simple separation process, easy in-vitro amplification, good biocompatibility, difficult allergy, good mechanical strength, no breakage and no substantial degradation.
Description
Technical Field
The invention relates to the technical field of medical materials, in particular to a scaffold composite material and a preparation method and application thereof.
Background
Mesenchymal Stem Cells (MSCs) are a heterogeneous group of cells derived from a matrix, and can be obtained from most tissues of the human body. They have self-renewal, tissue repair, immunoregulatory capabilities, and can differentiate towards the mesodermal lineage, for example: adipocytes, osteocytes, chondrocytes, and the like, and can also differentiate into cells of other germ layer lineages, for example, epidermal cells and vascular endothelial cells.
The stem cells have potential functions of regenerating various tissues and organs and human bodies by virtue of extremely strong self-replication capacity and differentiation capacity. Compared with other seed cells, the mesenchymal stem cells have the advantages of wide source, rich content, simple separation, stable and easy in vitro amplification, no ethical problem, convenience for autologous transplantation, low immunogenicity, small damage to supply areas and the like, and are highly valued by surgeons.
However, the existing scaffold material is poor in mechanical strength if being made of biological material, is easy to loosen in the clamping process of an operating forceps and after being added with a culture medium, and is easy to break and greatly degrade in the culture process.
disclosure of Invention
The invention aims to provide a scaffold composite material, and a preparation method and application thereof.
in one aspect, an embodiment of the present invention provides a method for preparing a scaffold composite material, where the method includes the following steps:
S1, preparing single cell suspension of the mesenchymal stem cells, wherein the concentration of the single cell suspension is 0.9 multiplied by 105To 1.1X 106Per ml;
S2, mixing the hydrolyzed placenta extract, the biological macromolecules, the degradable macromolecules and the solvent, and adjusting the pH value to 7.2-7.4 to prepare a first scaffold material premixed solution;
and S3, mixing the single cell suspension of the mesenchymal stem cells prepared in the S1 and the first scaffold material premixed solution prepared in the S2 according to a preset formula, and preparing the scaffold composite material.
As a further improvement of the embodiment of the present invention, before step S2, the method further comprises the step of preparing a hydrolyzed placenta extract in advance, specifically comprising the steps of:
1. Preparing a placenta tissue block;
washing red loose tissues and residual placenta tissues stripped during the separation of the mesenchymal stem cells with purified water to remove blood clots on the surfaces, adding DMEM (DMEM) to immerse the tissues, freezing the tissues at the temperature of-80 ℃, unfreezing the tissues in a water bath at the temperature of 37 ℃, washing the tissues with deionized water at the temperature of 4 ℃, eluting the tissues with 1% Triton x-100 and 0.1% ammonium hydroxide, washing the tissues with deionized water, washing the tissues with PBS and washing the tissues with the deionized water; draining the tissue mass;
2. Preparing freeze-dried placenta tissues with different particle sizes;
Placing the drained tissue in a refrigerator at-80 ℃ for freezing and freeze-drying for 24h, and crushing the freeze-dried tissue by using a high-speed crusher; screening with 100um, 70um and 4um cell sieves to obtain different particle sizes; repeatedly crushing placenta tissues with the particle sizes of 100um and 70 um;
3. preparing a hydrolyzed placenta extract;
Sieving with 4um cell sieve, adding 9mL of 0.1M HCl into per gram of placenta powder with particle size less than 4um, centrifuging at room temperature, adjusting pH of the supernatant to 7.4 with 1M NaOH, and filtering the supernatant with 0.22um filter to obtain hydrolyzed placenta extract.
As a further improvement of the embodiment of the present invention, the step S1 includes preparing a single cell suspension of mesenchymal stem cells, specifically including the following steps:
S101, separating and culturing mesenchymal stem cells;
S102, carrying out passage expansion on the mesenchymal stem cell;
s103, taking the 2 nd generation cells in the logarithmic phase, sucking out the culture medium, adding a digestive juice for digestion, stopping digestion with a proper amount of serum, and blowing to form a single cell suspension.
As a further improvement of the embodiment of the present invention, the step S101 of isolating and culturing the mesenchymal stem cells specifically comprises:
the placenta tissue of a healthy donor is taken and washed repeatedly by PBS for a plurality of times, and the amnion on the surface of the placenta is stripped. Taking small pieces of tissue, scraping off the attached red loose tissue with tiger's tooth forceps, retaining all blood vessels, cleaning with normal saline, and shearing placenta tissue into 1-4 mm2Homogenizing the tissue, digesting with collagenase, centrifuging the digestive juice for 10min, and filling the cell precipitate with serum-free and phenol red-free mediumculturing with special culture medium for cytoplasmic stem cell at 37 deg.C under CO2Culturing in an incubator with the volume fraction of 5 percent.
As a further improvement of the embodiment of the invention, the specific steps of subculturing, expanding and digesting the mesenchymal stem cell cells and forming the single cell suspension comprise:
When the cell confluence rate reaches 70%, carrying out passage, adding preheated 0.05% pancreatin, slightly rotating to cover the bottom of the bottle, and placing the bottle in a 37 ℃ incubator for digestion for 3-5 min; digesting for 3min, and observing under a microscope; most cells round and digestion is stopped when some cells have been suspended;
Washing the culture flask with DMEM after sucking out the cell suspension, centrifuging for 10min, sucking out the supernatant, resuspending the cell precipitate in the culture medium, counting, 3000 plus 6000 cells/cm2Inoculating into T75 culture flask at a density of 3000-6000 cells/cm, and performing secondary passage operation when the confluency of P1 cells reaches 70%2The density was inoculated into T175 flasks.
as a further improvement of the embodiments of the present invention, the biomacromolecule comprises chitin and collagen.
As a further improvement of the embodiment of the invention, the growth factor comprises any one or more of epidermal growth factor EGF, fibroblast growth factor FGF, keratinocyte growth factor KGF-2, platelet derived factor PDGF, transforming growth factor TGF-beta, vascular endothelial growth factor VEGF, hepatocyte growth factor HGF, insulin-like growth factor IGF, interleukin 6 and interleukin 8.
As a further refinement of an embodiment of the present invention, the collagen is type I collagen.
As a further improvement of an embodiment of the present invention, the mesenchymal stem cells are extracted from animals, allogenic or autologous, or extracted from placenta tissue, umbilical cord, bone marrow, adipose tissue.
As a further improvement of the embodiment of the present invention, the hydrolyzed placenta extract is extracted from placenta of animals or placenta of human xenogeneic, placenta produced by self-parturient.
in another aspect, the embodiment of the invention provides a scaffold composite material obtained by the preparation method.
In a further aspect, the embodiment of the invention provides the application of the scaffold composite material obtained by the preparation method in the preparation of a scaffold for wound repair.
Compared with the prior art, the invention has the following technical effects:
1. The embodiment of the invention utilizes the human stem cells, has strong regeneration, repair and differentiation capacities, can obviously promote the repair and regeneration of aging cells, has mesenchymal stem cell secretion, has more varieties and contents of bioactive substances, and the active factor EGF is matched with the synergistic action of FGF, KGF and the like, can support the repair and proliferation of damaged cells, provides a microenvironment for tissue regeneration and cell proliferation, helps the proliferation of dermal fibroblasts, and promotes the synthesis of collagen;
2. The stem cell secretion and the decellularized placenta tissue are applied in the embodiment of the invention, so that the risk of anaphylactic reaction possibly caused by directly using stem cells is reduced, and the problem of low cell survival rate is solved to a certain extent by utilizing the characteristics of a decellularized biological material with low immunogenicity, such as natural specific three-dimensional structure, rich active factors and the like; the scaffold material can also provide a proper living and differentiation environment for mesenchymal stem cells, and the wound repair effect can be improved by selecting a proper biological scaffold material;
3. the novel scaffold material formed by the invention by utilizing the technology of combining the mesenchymal stem cells and the scaffold has good mechanical strength, can be clamped by operating forceps, is not loose after being added into a culture medium, and is not broken and greatly degraded in the continuous culture process.
drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 is a graph of example elution tissue HE staining.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
example one
The embodiment of the invention provides a preparation method of a scaffold composite material, which comprises the following steps:
s1, preparing single cell suspension of the mesenchymal stem cells, wherein the concentration of the single cell suspension is 0.9 multiplied by 105to 1.1X 106Per ml;
S2, mixing the hydrolyzed placenta extract, the biological macromolecules, the degradable macromolecules and the solvent, and adjusting the pH value to 7.2-7.4 to prepare a first scaffold material premixed solution;
And S3, mixing the single cell suspension of the mesenchymal stem cells prepared in the S1 and the first scaffold material premixed solution prepared in the S2 according to a preset formula, and preparing the scaffold composite material.
Wherein before step S2, the method further comprises the step of preparing a hydrolyzed placenta extract in advance, and the method specifically comprises the following steps:
1. preparing a placenta tissue block;
Washing red loose tissues and residual placenta tissues stripped during the separation of mesenchymal stem cells with purified water to remove blood clots on the surfaces, adding DMEM (DMEM) to immerse the tissues, freezing the tissues at the temperature of minus 80 ℃, then carrying out water bath at the temperature of 37 ℃ for 20min, repeating the steps for many times, adding the purified water, placing the tissues on a shaking table at the temperature of 4 ℃ and shaking the tissues for 24h, changing deionized water every 6h, eluting the tissues for 3 days by using 1% Triton x-100 and 0.1% ammonium hydroxide, and washing the tissues for 1 day by using the deionized water; washing the tissue mass with PBS for 1 day, and washing the tissue mass with deionized water for 1 day; draining the tissue blocks, and weighing and recording;
2. preparing freeze-dried placenta tissues with different particle sizes;
placing the drained tissue in a refrigerator at minus 80 ℃ for 24 hours, freeze-drying the frozen tissue for 24 hours, and crushing the freeze-dried tissue by using a high-speed crusher; screening with 100um, 70um and 4um cell sieves to obtain different particle sizes; repeatedly crushing placenta tissues with the particle sizes of 100um and 70 um;
3. preparing a hydrolyzed placenta extract;
sieving with 4um cell sieve, adding 9mL of 0.1M HCl into placenta powder with particle size less than 4um per gram, shaking at room temperature (26 deg.C) for 48h at 1500rpm for 15min, centrifuging for 3 times, adding 40mL deionized water into the supernatant precipitate, mixing, centrifuging again to obtain supernatant, adjusting pH to 7.4 with 1M NaOH, and filtering with 0.22um filter to obtain hydrolyzed placenta extract.
After the step 3, DNA is extracted by using a Tiangen DP304 kit, the DNA content of 100g of fresh placenta tissue is about 1789.2ng/mg, the average DNA content of 9.1g of freeze-dried powder after decellularization is 44.6ng/mg, and the decellularization efficiency is 99.8 percent; the HE staining section result of the tissue after decellularization is shown in figure 1, and the cell nucleus distribution is not obviously visible in the figure, which indicates that the decellularization effect meets the requirement and the allergen is effectively removed.
Wherein, in the embodiment of the invention, the decellularization efficiency of the hydrolyzed placenta extract from placenta tissues reaches 99-99.9%.
further, step S1 includes preparing a single cell suspension of the mesenchymal stem cells, specifically including the following steps:
S101, separating and culturing mesenchymal stem cells;
S102, carrying out passage expansion on the mesenchymal stem cell;
S103, taking the 2 nd generation cells in the logarithmic phase, sucking out the culture medium, adding a digestive juice for digestion, stopping digestion with a proper amount of serum, and blowing to form a single cell suspension.
wherein, the step S101 of separating and culturing the mesenchymal stem cells comprises the following specific steps:
The placenta tissue of a healthy donor is taken and washed repeatedly by PBS for a plurality of times, and the amnion on the surface of the placenta is stripped. Collecting small pieces of tissue, scraping off the red loose tissue with tiger tooth forceps, and retainingcleaning all blood vessels with normal saline, and shearing placenta tissue into 1-4 mm2Homogenizing the tissue, digesting with collagenase, centrifuging the digestive juice for 10min, culturing the cell precipitate with serum-free and phenol red-free special culture medium for mesenchymal stem cells at 37 deg.C and CO2Culturing in an incubator with the volume fraction of 5 percent.
As a further improvement of the embodiment of the invention, the specific steps of subculturing, expanding and digesting the mesenchymal stem cell cells and forming the single cell suspension comprise:
When the cell confluence rate reaches 70%, carrying out passage, adding preheated 0.05% pancreatin, slightly rotating to cover the bottom of the bottle, and placing the bottle in a 37 ℃ incubator for digestion for 3-5 min; digesting for 3min, and observing under a microscope; most cells round and digestion is stopped when some cells have been suspended;
washing the culture flask with DMEM after sucking out the cell suspension, centrifuging for 10min, sucking out the supernatant, resuspending the cell precipitate in the culture medium, counting, 3000 plus 6000 cells/cm2Inoculating into T75 culture flask at a density of 3000-6000 cells/cm, and performing secondary passage operation when the confluency of P1 cells reaches 70%2The density was inoculated into T175 flasks.
in an embodiment of the invention, the biomacromolecule is chitin; the growth factor comprises any one or more of epidermal growth factor EGF, fibroblast growth factor FGF, keratinocyte growth factor KGF-2, platelet derived factor PDGF, transforming growth factor TGF-beta, vascular endothelial growth factor VEGF, hepatocyte growth factor HGF, insulin-like growth factor IGF, interleukin 6 and interleukin 8.
The growth factors of the embodiment of the invention adopt various compounds, and the content measurement result is shown in the table I:
Table contents of various active factors are
Specifically, the collagen is type I collagen.
In embodiments of the invention, the mesenchymal stem cells are extracted from animals, allogenic or autologous, or from placental tissue, umbilical cord, bone marrow, adipose tissue, while the hydrolyzed placental extract is extracted from the placenta of animals or from the placenta of human allogenic, the placenta resulting from the parturient's own childbirth.
in the embodiment of the present invention, the following ratio may be selected from a preset formula of the single cell suspension of the mesenchymal stem cells prepared in S1 and the first scaffold material premix solution prepared in S2 mixed according to the preset formula in step S3:
Example 1
Mesenchymal stem cell suspension 0.9 × 1051.1X 10 per ml5Per ml
3 to 5.5 percent of hydrolyzed placenta extract
chitin 1.5-2.5%
0.4 to 0.6 percent of type I collagen
EGF 4.5～5.5ng/ml
example 2
Mesenchymal stem cell suspension 0.9 × 1051.1X 10 per ml5Per ml
2 to 3.5 percent of hydrolyzed placenta extract
Chitin 1.8-2.2%
0.4 to 0.6 percent of type I collagen
EGF 4.5～5.5ng/ml
example 3
mesenchymal stem cell suspension 1.0 × 1051.1X 10 per ml5Per ml
3 to 5.5 percent of hydrolyzed placenta extract
Chitin 2.0-2.5%
0.4 to 0.6 percent of type I collagen
EGF 4.5～5.5ng/ml
Example two
the embodiment of the present invention provides a scaffold composite material obtained by the preparation method disclosed in the first embodiment, and specific examples also refer to the composition in the mixture ratio in the first embodiment 1, and it is to be noted that:
in the embodiment of the invention, the collagen can also be selected from other materials A, the material A is selected from acellular matrix and one or more of type II collagen, silk fibroin, chitin and derivatives thereof, hyaluronic acid, alginic acid, gelatin and fibrin;
In the embodiment of the present invention, a material B may be further added, where the material B is selected from any one or a combination of several of polycaprolactone, hydroxyapatite (nano-hydroxyapatite), polylactic acid, polyglycolic acid, lactic acid-glycolic acid copolymer, polymethyl methacrylate, octacalcium phosphate, tricalcium phosphate, polydioxanone, PVA, polyurethane, polycarbonate, polyhydroxyalkanoates (polyhydroxybutyrate and polyhydroxyvalerate and copolymers), polysaccharides, polyethylene oxide, polypropylene oxide, polyglycolide-co-trimethylene carbonate, polymer sugar, proteins, hydrophilic block copolymers, polyuronic acid, mucopolysaccharides, polyethylene oxide, surfactants, polyhydroxy compounds, polyhydroxy esters, fatty alcohols, fatty alcohol esters, fatty acids, fatty acid esters, and liquid silicone; other auxiliary agents, such as cellulose and glass for binding, metal, growth hormone, protein, polyvinyl alcohol, fumaric acid, hyaluronic acid, citric acid, etc.;
the embodiment of the invention comprises that the mesenchymal stem cell suspension, hydrolyzed placenta extract and other biological macromolecules, including but not limited to the traditional cartilage scaffold materials such as the material A and the material B mentioned above;
traditional stent materials also include the following formulations:
Conventional stents include hydrogel a and hydrogel B; the using mass ratio of the hydrogel A to the hydrogel B in printing is 4: 3-3: 2;
wherein, hydrogel A: 40-50 parts of methacryloyl glycinamide hydrogel, 10-15 parts of monetite, 13-18 parts of calcium sulfate, 0.3-0.6 part of nonionic surfactant and 16.4-36.7 parts of ethyl acetate;
In hydrogel B: 22-30 parts of silicon dioxide gel, 18-26 parts of silicic acid, 25-32 parts of polylactic acid short fiber, 0.2-0.5 part of zwitterionic surfactant and 11.5-34.8 parts of ethanol.
The formula of the slurry of the traditional bracket II comprises 4-8% of polyvinyl alcohol aqueous solution, copper oxide and a calcium-phosphorus-containing biological ceramic material, wherein the mass-volume ratio of the calcium-phosphorus-containing biological ceramic material to the polyvinyl alcohol aqueous solution is 2.5-3.5g/mL, and the mass ratio of the copper oxide to the calcium-phosphorus-containing biological ceramic material is 0.005-0.05: 1. Wherein the biological ceramic material containing calcium and phosphorus is hydroxyapatite, octacalcium phosphate, calcium phosphate or biphase calcium phosphate, and the particle size is 10-15 μm.
the formula of the traditional stent III comprises type II collagen, hyaluronic acid, chondroitin sulfate and hydroxyapatite; wherein the extracted type II collagen is prepared into a type II collagen solution with the concentration of 1-2% (m/V), the uniform mixed solution system is transferred into a 24-hole plate, and the three-dimensional cartilage scaffold is obtained after vacuum freeze drying.
EXAMPLE III
the embodiment of the invention provides application of the scaffold composite material obtained by the preparation method in manufacturing a scaffold for wound repair.
the specific preparation process of the bracket comprises the following steps:
In the embodiment of the invention, the prepared single cell suspension of the mesenchymal stem cells and the scaffold composite material prepared by mixing the single cell suspension of the mesenchymal stem cells, the hydrolyzed placenta extract, the biological macromolecules, the collagen and the growth factors in the embodiment 2 are printed, and the scaffold composite material in the embodiment 2 is used as a scaffold structure and is printed into a pore-containing structure which is orderly arranged, wherein the pore diameter is 100-500 mu m, and the pore structure can be an orthogonal square hole or a triangular hole.
the loaded cells were printed and filled in the pores of the scaffold composite described in example 2, stacked layer by layer, and cross-linked cultured after printing.
Further, printing can be performed by 3D printing equipment, printing a pore structure with an ordered arrangement, and arranging cells according to 1x105-1x107per ml, the printed scaffold composite scaffold described in example 2 was soaked in water and cross-linked to obtain a bone scaffold.
Printing the scaffold composite material of embodiment 2 by using 3D printing equipment, printing a pore structure with ordered arrangement, soaking the printed scaffold composite material of embodiment 2 in the material A, and freeze-drying for 12-24h after crosslinking to obtain the scaffold.
in order to further verify the application of the scaffold composite material in the preparation of a scaffold for wound repair, the scaffold composite material prepared by the invention is tested for mechanical properties and biocompatibility, and the experimental design and parameters are as follows:
1. Cell viability assay:
Placing the prepared composite material bracket loaded with the mesenchymal stem cells into a culture medium for culturing, staining the cells in the composite material bracket with a live/dead cell staining kit when culturing for 1, 4 and 7 days, observing and taking a picture under a fluorescent microscope, counting the number of the live cells and the number of the dead cells in the bracket respectively, and calculating the percentage of the live cells in the total number of the cells; the result shows that the cell survival rate is more than 80%, which indicates that the prepared scaffold composite material can well maintain the cell activity and has good cell compatibility.
2. Aperture detection
drying the prepared composite material bracket, spraying gold on the surface, placing the composite material bracket under a scanning electron microscope for surface morphology observation, and taking a picture for recording; the pore size and the network distribution of the composite material scaffold are mainly observed, and the measurement result shows that the pore size of the composite material scaffold disclosed by the embodiment of the invention is 100-500um, the pores are communicated with each other, the specific surface area is improved, the transportation of oxygen and nutrient substances and the removal of metabolic substances are facilitated, and the adhesion, growth and proliferation of cells are facilitated.
3. Mechanical property detection
Detecting the compressive strength of the composite material bracket by using a universal testing machine, measuring the height and the diameter of the composite material bracket, keeping the compression speed of 1mm/min, drawing a stress-strain curve according to measured data, and calculating the compressive strength; the test result shows that the formed new composite material bracket has better mechanical strength, can be clamped by an operating forceps, is not loose after being added into a culture medium, and is not broken and greatly degraded in the continuous culture process.
compared with the prior art, the invention has the following technical effects:
1. the embodiment of the invention utilizes the human stem cells, has strong regeneration, repair and differentiation capacities, can obviously promote the repair and regeneration of aging cells, has mesenchymal stem cell secretion, has more varieties and contents of bioactive substances, and the active factor EGF is matched with the synergistic action of FGF, KGF and the like, can support the repair and proliferation of damaged cells, provides a microenvironment for tissue regeneration and cell proliferation, helps the proliferation of dermal fibroblasts, and promotes the synthesis of collagen;
2. The stem cell secretion and the decellularized placenta tissue are applied in the embodiment of the invention, so that the risk of anaphylactic reaction possibly caused by directly using stem cells is reduced, and the problem of low cell survival rate is solved to a certain extent by utilizing the characteristics of a decellularized biological material with low immunogenicity, such as natural specific three-dimensional structure, rich active factors and the like; the scaffold material can also provide a proper living and differentiation environment for mesenchymal stem cells, and the wound repair effect can be improved by selecting a proper biological scaffold material;
3. The novel scaffold material formed by the invention by utilizing the technology of combining the mesenchymal stem cells and the scaffold has good mechanical strength, can be clamped by operating forceps, is not loose after being added into a culture medium, and is not broken and greatly degraded in the continuous culture process.
In the foregoing description of embodiments, the particular features, structures, materials, or characteristics may be combined in any suitable manner in any one or more embodiments or examples.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (10)
1. a preparation method of a scaffold composite material is characterized by comprising the following steps:
S1, preparing single cell suspension of the mesenchymal stem cells, wherein the concentration of the single cell suspension is 0.9 multiplied by 105to 1.1X 106Per ml;
s2, mixing the hydrolyzed placenta extract, the biological macromolecules, the degradable macromolecules and the solvent, and adjusting the pH value to 7.2-7.4 to prepare a first scaffold material premixed solution;
And S3, mixing the single cell suspension of the mesenchymal stem cells prepared in the S1 and the first scaffold material premixed solution prepared in the S2 according to a preset formula, and preparing the scaffold composite material.
2. The method for preparing the scaffold composite material according to claim 1, wherein the step S2 is preceded by preparing a hydrolyzed placenta extract in advance, and the method specifically comprises the following steps:
(1) Preparing a placenta tissue block;
washing red loose tissues and residual placenta tissues stripped during the separation of the mesenchymal stem cells with purified water to remove blood clots on the surfaces, adding DMEM (DMEM) to immerse the tissues, freezing the tissues at the temperature of-80 ℃, unfreezing the tissues in a water bath at the temperature of 37 ℃, washing the tissues with deionized water at the temperature of 4 ℃, eluting the tissues with 1% Triton x-100 and 0.1% ammonium hydroxide, washing the tissues with deionized water, washing the tissues with PBS and washing the tissues with the deionized water; draining the tissue mass;
(2) preparing freeze-dried placenta tissues with different particle sizes;
Placing the drained tissue in a refrigerator at-80 ℃ for freezing and freeze-drying for 24h, and crushing the freeze-dried tissue by using a high-speed crusher; screening with 100um, 70um and 4um cell sieves to obtain different particle sizes; repeatedly crushing placenta tissues with the particle sizes of 100um and 70 um;
(3) Preparing a hydrolyzed placenta extract;
sieving with 4um cell sieve, adding 9mL of 0.1M HCl into per gram of placenta powder with particle size less than 4um, centrifuging at room temperature, adjusting pH of the supernatant to 7.4 with 1M NaOH, and filtering the supernatant with 0.22um filter to obtain hydrolyzed placenta extract.
3. The method for preparing the scaffold composite material according to claim 1, wherein the step S1 comprises preparing a single cell suspension of mesenchymal stem cells, and specifically comprises the following steps:
S101, separating and culturing mesenchymal stem cells;
S102, carrying out passage expansion on the mesenchymal stem cell;
s103, taking the 2 nd generation cells in the logarithmic phase, sucking out the culture medium, adding a digestive juice for digestion, stopping digestion with a proper amount of serum, and blowing to form a single cell suspension.
4. the method for preparing the scaffold composite material according to claim 3, wherein the step S101 of isolating and culturing mesenchymal stem cells comprises the following steps:
The placenta tissue of a healthy donor is taken and washed repeatedly by PBS for a plurality of times, and the amnion on the surface of the placenta is stripped. Taking small pieces of tissue, scraping off the attached red loose tissue with tiger's tooth forceps, retaining all blood vessels, cleaning with normal saline, and shearing placenta tissue into 1-4 mm2Homogenizing the tissue, digesting with collagenase, centrifuging the digestive juice for 10min, culturing the cell precipitate with serum-free and phenol red-free special culture medium for mesenchymal stem cells at 37 deg.C and CO2Culturing in an incubator with the volume fraction of 5 percent.
5. the preparation method of the scaffold composite material according to claim 3, wherein the specific steps of subculturing, expanding and digesting the mesenchymal stem cell cells to form a single cell suspension comprise:
when the cell confluence rate reaches 70%, carrying out passage, adding preheated 0.05% pancreatin, slightly rotating to cover the bottom of the bottle, and placing the bottle in a 37 ℃ incubator for digestion for 3-5 min; digesting for 3min, and observing under a microscope; most cells round and digestion is stopped when some cells have been suspended;
washing the culture flask with DMEM after sucking out the cell suspension, centrifuging for 10min, sucking out the supernatant, resuspending the cell precipitate in the culture medium, counting, 3000 plus 6000 cells/cm2Inoculating into T75 culture flask at a density of 3000-6000 cells/cm, and performing secondary passage operation when the confluency of P1 cells reaches 70%2The density was inoculated into T175 flasks.
6. the method of preparing a scaffold composite according to claim 1, wherein the biomacromolecules comprise chitin, collagen;
the growth factor comprises any one or a combination of more of epidermal growth factor EGF, fibroblast growth factor FGF, keratinocyte growth factor KGF-2, platelet derived factor PDGF, transforming growth factor TGF-beta, vascular endothelial growth factor VEGF, hepatocyte growth factor HGF, insulin-like growth factor IGF, interleukin 6 and interleukin 8;
the collagen is type I collagen.
7. The method of preparing a scaffold composite according to claim 1, wherein said mesenchymal stem cells are extracted from animals, allo-or autologous, or from placental tissue, umbilical cord, bone marrow, adipose tissue.
8. The method of preparing a scaffold composite according to claim 1, wherein said hydrolyzed placental extract is extracted from the placenta of an animal or human xenogeneic, the placenta resulting from the spontaneous delivery of a parturient.
9. The scaffold composite obtained by the preparation method according to any one of claims 1 to 8.
10. Use of the scaffold composite obtained by the preparation method according to any one of claims 1 to 8 in the manufacture of a scaffold for wound repair.
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