CN115607729B - Biological ink, 3D printing hydrogel bracket and preparation method and application - Google Patents

Biological ink, 3D printing hydrogel bracket and preparation method and application Download PDF

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CN115607729B
CN115607729B CN202211353364.9A CN202211353364A CN115607729B CN 115607729 B CN115607729 B CN 115607729B CN 202211353364 A CN202211353364 A CN 202211353364A CN 115607729 B CN115607729 B CN 115607729B
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printing
hydrogel
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berberine
mixture
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CN115607729A (en
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王东阳
张一迪
聂然
周延民
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Jilin University
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Abstract

The invention provides a biological ink, a 3D printing hydrogel bracket, a preparation method and application thereof, and belongs to the technical field of bone tissue engineering materials. The biological ink provided by the invention comprises agarose, alginate, carboxymethyl chitosan, hydroxyapatite, berberine, bone marrow mesenchymal stem cells and PBS buffer solution. The 3D printing hydrogel scaffold prepared by using the biological ink provided by the invention has good safety and biocompatibility, no immunological rejection reaction, and the berberine can promote osteogenic differentiation of bone marrow mesenchymal stem cells, so that the 3D printing hydrogel scaffold provided by the invention can be used as a bone repair regeneration material in bone tissue engineering.

Description

Biological ink, 3D printing hydrogel bracket and preparation method and application
Technical Field
The invention relates to the technical field of bone tissue engineering materials, in particular to a biological ink, a 3D printing hydrogel bracket, a preparation method and application.
Background
The 3D biological printing technology is developed rapidly in the field of bone tissue engineering, and is a rapid molding method for realizing bionic, accurate printing and personalized scaffold structure by using cells and biological materials as biological ink, and the size, shape and porosity of the scaffold can be designed at will.
Bone defects or bone injuries caused by trauma, tumors, degenerative diseases, surgery, etc. can have serious physiological and psychological effects on patients. At present, a plurality of bone injury therapies are clinically applied to implant autologous bone or allogeneic bone. Autologous bone grafting is extracted from normal bone tissue in a patient, but because of limited autologous bone sources, a great deal of clinical references cannot be met, and the application universality of the autologous bone grafting is limited. When the allogeneic bone is used for bone implantation, although the source problem does not exist, the allogeneic bone in the prior art easily causes host to generate immune rejection reaction, so that the allogeneic bone implantation is easy to fail.
Disclosure of Invention
The invention aims to provide the bio-ink, the 3D printing hydrogel stent, the preparation method and the application, and all the components in the bio-ink are nontoxic and have no side effect, and the 3D printing hydrogel stent prepared by using the bio-ink is good in safety and biocompatibility, free from immune rejection reaction and can be used as a bone repair regeneration material in bone tissue engineering.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a biological ink, which comprises agarose, alginate, carboxymethyl chitosan, hydroxyapatite, berberine, bone marrow mesenchymal stem cells and PBS buffer solution; the concentration of agarose in the biological ink is 0.005-0.025 g/mL, the concentration of alginate is 0.03-0.07 g/mL, the concentration of carboxymethyl chitosan is 0.03-0.07 g/mL, the concentration of hydroxyapatite is 0.003-0.007 g/mL, the concentration of berberine is 0.005-0.02 g/mL, and the concentration of bone marrow mesenchymal stem cells is 1X 10 7 ～5×10 7 Individual cells/mL.
Preferably, the pH of the PBS buffer is 7.4.
The invention provides a preparation method of the biological ink, which comprises the following steps:
dissolving agarose in PBS buffer solution to obtain a first mixture;
mixing the first mixture with an alginate to obtain a second mixture;
mixing the second mixture, carboxymethyl chitosan and hydroxyapatite to obtain a third mixture;
and mixing the third mixture, berberine and bone marrow mesenchymal stem cells to obtain the biological ink.
The invention provides a preparation method of a 3D printing hydrogel bracket, which comprises the following steps:
3D bio-printing is carried out on the bio-ink according to the technical scheme or the bio-ink prepared by the preparation method according to the technical scheme, so as to obtain a 3D printing bracket;
immersing the 3D printing support in soluble calcium salt water solution for crosslinking treatment to obtain the 3D printing hydrogel support.
Preferably, the diameter of the nozzle used for 3D bio-printing is 100-1000 μm.
Preferably, the printing temperature of the 3D biological printing is 37 ℃, and the cooling plate is precooled to 4-10 ℃.
Preferably, the printing parameters of the 3D bioprinting include: each layer has a thickness of 0.05-0.5 mm, a layer number of 5-20 layers, a line spacing of 0.2-2 mm and a printing speed of 5-20 mm/s.
Preferably, ca in the aqueous solution of soluble calcium salt 2+ The concentration of (2) is 0.01-0.03 g/mL; the temperature of the crosslinking treatment is 25-37 ℃ and the time is 15-20 min.
The invention provides a 3D printing hydrogel stent prepared by the preparation method, which comprises a hydrogel matrix, and hydroxyapatite, berberine and bone marrow mesenchymal stem cells loaded in the hydrogel matrix, wherein the hydrogel matrix is prepared from agarose, alginate and carboxymethyl chitosan in Ca 2+ Crosslinking is formed under the action of the catalyst.
The invention provides application of the 3D printing hydrogel scaffold in bone tissue engineering.
The invention provides a biological ink, which comprises agarose, alginate, carboxymethyl chitosan, hydroxyapatite, berberine, bone marrow mesenchymal stem cells and PBS buffer solution; the concentration of agarose in the biological ink is 0.005-0.025 g/mL, the concentration of alginate is 0.03-0.07 g/mL, the concentration of carboxymethyl chitosan is 0.03-0.07 g/mL, the concentration of hydroxyapatite is 0.003-0.007 g/mL, the concentration of berberine is 0.005-0.02 g/mL, and the concentration of bone marrow mesenchymal stem cells is 1X 10 7 ～5×10 7 Individual cells/mL. The 3D printing hydrogel scaffold prepared by using the biological ink provided by the invention has good safety and biocompatibility, no immunological rejection reaction, and the berberine can promote osteogenic differentiation of bone marrow mesenchymal stem cells, so that the 3D printing hydrogel scaffold provided by the invention can be used as a bone repair regeneration material in bone tissue engineering.
Drawings
FIG. 1 is a flow chart of the invention for preparing and performance testing a 3D printed hydrogel scaffold;
fig. 2 is a schematic view and a physical view of the 3D printing support prepared in example 1;
FIG. 3 is a graph showing the compressive strength results of the 3D printed hydrogel scaffold prepared in example 1;
FIG. 4 is a graph showing the swelling ratio results of the 3D-printed hydrogel scaffold prepared in example 1;
FIG. 5 is an SEM image of the freeze-dried scaffolds obtained after freeze-drying of the 3D printed hydrogel scaffolds prepared in example 1 and comparative examples 1-3;
FIG. 6 is a graph showing the degradation rate results of the 3D printing hydrogel scaffold prepared in example 1;
FIG. 7 is a graph showing the results of in vitro biocompatibility tests of 3D printed hydrogel scaffolds prepared in example 1 and comparative example 3;
FIG. 8 is a release profile of berberine in the 3D printed hydrogel scaffold prepared in example 1;
FIG. 9 is a graph showing the results of ALP activity tests of BMSCs in 3D-printed hydrogel scaffolds prepared in example 1 and comparative example 3 after 7 and 14 days from the induction of bone formation;
FIG. 10 is a Micro CT 3D image of the rat skull after implantation of the 3D printed hydrogel scaffolds prepared in example 1 and comparative examples 1-3;
FIG. 11 is a graph showing the results of measuring the formation volume of new bone at the skull defect site of a rat after implantation of the 3D-printed hydrogel scaffolds prepared in example 1 and comparative examples 1 to 3
FIG. 12 is a graph showing the coloration of rat skull HE after implantation of the 3D printing hydrogel scaffolds prepared in example 1 and comparative examples 1 to 3;
FIG. 13 is a chart showing the HE staining of visceral tissues of rats after implantation of the 3D-printed hydrogel scaffolds prepared in example 1 and comparative examples 1 to 3.
Detailed Description
The invention provides a biological ink, which comprises agarose, alginate, carboxymethyl chitosan, hydroxyapatite, berberine, bone marrow mesenchymal stem cells and PBS buffer solution. In the invention, the concentration of agarose in the biological ink is 0.005-0.025 g/mL, preferably 0.015g/mL; the concentration of the alginate is 0.03-0.07 g/mL, preferably 0.05g/mL, and the alginate is preferably alginic acidSodium; the concentration of the carboxymethyl chitosan is 0.03-0.07 g/mL, preferably 0.05g/mL; the concentration of the hydroxyapatite is 0.003-0.007 g/mL, preferably 0.005g/mL; the concentration of berberine is 0.005-0.02 g/mL, preferably 0.01g/mL; the concentration of the mesenchymal stem cells is 1×10 7 ～5×10 7 Individual cells/mL, preferably 10 7 Individual cells/mL. In the present invention, the pH of the PBS buffer solution is preferably 7.4.
In the invention, agarose has good biocompatibility, high melting point and good rheological property, and is a good carrier material; alginate is an anionic polysaccharide with high biocompatibility, mild gel forming performance and low price; carboxymethyl chitosan is a polymeric polysaccharide with good biodegradability and biocompatibility, and the degradation products can support the growth of cells and tissues; the invention adopts agarose, alginate and carboxymethyl chitosan as matrix materials to prepare hydrogel with a cross-linked three-dimensional network structure, has good biocompatibility, is a good slow release carrier, can release drug molecules loaded in the hydrogel, and can maintain the concentration of the drug in the environment. In the invention, berberine is isoquinoline alkaloid which can be extracted from various herbaceous plants, and has antibacterial effect, and pharmacological effects such as anti-tumor and anti-inflammatory effects; the bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMSCs) are cell subsets with various differentiation potential found in bone marrow stroma, can differentiate into bone, cartilage, fat, nerve and myoblast cells, and the BMSCs have the characteristics of strong in vitro expansion capability, low immunogenicity and the like, are ideal choices of bone regeneration engineering, and the presence of berberine can promote the osteogenic differentiation of the BMSCs.
The invention provides a preparation method of the biological ink, which comprises the following steps:
dissolving agarose in PBS buffer solution to obtain a first mixture;
mixing the first mixture with an alginate to obtain a second mixture;
mixing the second mixture, carboxymethyl chitosan and hydroxyapatite to obtain a third mixture;
and mixing the third mixture, berberine and bone marrow mesenchymal stem cells to obtain the biological ink.
Agarose is dissolved in PBS buffer solution to obtain a first mixture. In the present invention, the dissolution is preferably performed under microwave heating conditions, the present invention preferably employs microwave heating to boiling, and then stops the microwave heating, shaking every 5s, and then repeats the above-described process until complete dissolution, and cools to room temperature (25 ℃) to obtain a first mixture.
After obtaining the first mixture, the invention mixes the first mixture with alginate to obtain the second mixture. In the present invention, the mixing of the first mixture with the alginate is preferably performed under heating conditions, the temperature of the heating is preferably 55 to 65 ℃, more preferably 60 ℃; the heating time is preferably 25 to 35min, more preferably 30min; stirring is preferably continued during the heating.
After the second mixture is obtained, the second mixture, carboxymethyl chitosan and hydroxyapatite are mixed to obtain a third mixture. In the present invention, the mixing of the second mixture, carboxymethyl chitosan and hydroxyapatite is preferably performed under heating conditions, and the heating temperature is preferably 55 to 65 ℃, more preferably 60 ℃; the heating time is preferably 50 to 70min, more preferably 60min; preferably stirring continuously during the heating process; after the heating was completed, it was cooled to room temperature to obtain a third mixture.
After the third mixture is obtained, the third mixture, berberine and bone marrow mesenchymal stem cells are mixed to obtain the biological ink. The method for mixing the third mixture, the berberine and the bone marrow mesenchymal stem cells is not particularly limited, and the components are fully mixed.
The invention provides a preparation method of a 3D printing hydrogel bracket, which comprises the following steps:
3D bio-printing is carried out on the bio-ink according to the technical scheme or the bio-ink prepared by the preparation method according to the technical scheme, so as to obtain a 3D printing bracket;
immersing the 3D printing support in soluble calcium salt water solution for crosslinking treatment to obtain the 3D printing hydrogel support.
The 3D printing bracket is obtained by performing 3D biological printing on the biological ink. In the present invention, the diameter of the nozzle used for 3D bio-printing is preferably 100 to 1000 μm, more preferably 200 μm. The present invention preferably loads the bio-ink into a 5mL print cartridge and then performs 3D bio-printing through the above-mentioned diameter nozzle. In the present invention, the printing temperature of the 3D bioprinting is preferably 37 ℃; the present invention preferably limits the printing temperature to the above range, which is advantageous for smoothly pressing the bio-ink for 3D bio-printing. In the present invention, the cooling plate is preferably pre-cooled to 4-10 ℃, more preferably to 4 ℃ before the 3D bioprinting. In the present invention, the printing parameters of the 3D bioprinting include: the thickness of each layer is preferably 0.05 to 0.5mm, more preferably 0.15mm; the number of layers is preferably 5 to 20, more preferably 10; the line spacing is preferably 0.2 to 2mm, more preferably 1mm; the printing speed is preferably 5 to 20mm/s, more preferably 10mm/s. In the present invention, the overall size of the 3D printing support is preferably length×width×height=10×10×1.5mm (in vitro) or 5×5×1.5mm (in vivo).
After the 3D printing support is obtained, the 3D printing support is immersed in a soluble calcium salt water solution for crosslinking treatment, and the 3D printing hydrogel support is obtained. In the present invention, ca in the aqueous solution of soluble calcium salt 2+ The concentration of (2) is preferably 0.01 to 0.03g/mL, more preferably 0.02g/mL; the soluble calcium salt in the soluble calcium salt aqueous solution is preferably CaCl 2 . In the present invention, the temperature of the crosslinking treatment is preferably 25 to 37 ℃, more preferably room temperature; the time is preferably 15 to 20 minutes, more preferably 20 minutes. In the present invention, in Ca 2+ Under the action, agarose, alginate and carboxymethyl chitosan are crosslinked to form a hydrogel matrix, and hydroxyapatite, berberine and bone marrow mesenchymal stem cells are loaded in the hydrogel matrix, so that the 3D printing hydrogel bracket with stable morphology and structure is finally obtained.
The invention provides the technical prescriptionThe 3D printing hydrogel scaffold prepared by the preparation method comprises a hydrogel matrix, and hydroxyapatite, berberine and bone marrow mesenchymal stem cells loaded in the hydrogel matrix, wherein the hydrogel matrix is prepared from agarose, alginate and carboxymethyl chitosan in Ca 2+ Crosslinking is formed under the action of the catalyst. The 3D printing hydrogel stent provided by the invention has good physical properties and biocompatibility, wherein bone marrow mesenchymal stem cells can survive well in the stent, meanwhile, the stent can continuously release berberine to maintain the local drug concentration of the stent, the existence of the berberine can promote the osteogenic differentiation of the bone marrow mesenchymal stem cells in the stent, the good osteogenic effect of the stent is ensured, and good early-stage experimental foundation and theoretical support are provided for clinically treating bone defect patients.
The invention provides application of the 3D printing hydrogel scaffold in bone tissue engineering.
In the present invention, the raw materials used are commercially available products well known to those skilled in the art unless specified otherwise.
FIG. 1 is a flow chart of preparing a 3D printing hydrogel scaffold and performing performance test on the 3D printing hydrogel scaffold, specifically, agarose, alginate (specifically sodium alginate), carboxymethyl chitosan, hydroxyapatite, berberine, bone marrow mesenchymal stem cells and PBS buffer solution are firstly adopted to prepare biological ink, then 3D printing is performed, and the obtained 3D printing scaffold is immersed in soluble calcium salt water solution for crosslinking treatment, so that the 3D printing hydrogel scaffold is obtained; the bone marrow mesenchymal stem cells in the 3D printing hydrogel scaffold are differentiated into osteoblasts under the action of berberine, and the in-vitro osteogenesis capacity detection result is good; implanting the 3D printing hydrogel stent into a rat skull defect model, wherein a Micro-CT detection result shows that the 3D printing hydrogel stent has good in-vivo osteogenesis effect; after HE staining observation test, the rat skull tissue shows that the 3D printing hydrogel bracket has good in-vivo osteogenesis effect, and a large amount of new bone tissue is generated at the broken end.
The technical solution of the present invention will be clearly and completely described below with reference to an embodiment of the present invention and fig. 1. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Adding agarose (Ag) powder to PBS buffer solution (ph=7.4) under aseptic condition, heating to boiling using a microwave oven, then stopping heating of the microwave oven, shaking every 5s, repeating the above process until completely dissolved, and then cooling to room temperature (25 ℃) to obtain a first mixture; adding Sodium Alginate (SA) into the first mixture, and stirring and mixing for 30min at 60 ℃ to obtain a second mixture; adding carboxymethyl chitosan (CMC) and hydroxyapatite (Ha) into the second mixture, stirring and mixing for 1h at 60 ℃, and then cooling to room temperature to obtain a third mixture; adding berberine (Ber) and bone marrow mesenchymal stem cells (BMSCs) into the third mixture to obtain 3D biological printing ink; the concentration of agarose in the 3D biological printing ink is 0.015g/mL (marked as 1.5% w/v), the concentration of sodium alginate is 0.05g/mL (marked as 5% w/v), the concentration of carboxymethyl chitosan is 0.05g/mL (marked as 5% w/v), the concentration of hydroxyapatite is 0.005g/mL (marked as 0.5% w/v), the concentration of berberine is 0.01g/mL (marked as 1% w/v), and the concentration of mesenchymal stem cells is 10 7 Individual cells/mL;
loading the 3D bioprinting ink into a 5mL printing cylinder, and performing 3D bioprinting through a nozzle with the diameter of 200 μl to obtain a 3D printing bracket (shown in fig. 2), wherein the overall dimension of the 3D printing bracket is length×width×height=10×10×1.5mm (in vitro) or 5×5×1.5mm (in vivo); wherein, the in-process printing parameter of 3D bio-printing sets up as: the layer thickness is 0.15mm, the layer number is 10, the line spacing is 1mm, and the printing speed is 10mm/s; the printing temperature is set at 37 ℃ so as to smoothly squeeze the 3D bio-printing ink; precooling the cooling plate to 4 ℃;
immersing the 3D printing bracket in CaCl with the concentration of 0.02g/mL 2 In the aqueous solution, the crosslinking treatment is carried out for 20min under the room temperature condition,a 3D printed hydrogel scaffold was obtained, noted sample BC.
Comparative example 1
A 3D printed hydrogel scaffold was prepared according to the method of example 1, except that berberine and bone marrow mesenchymal stem cells were omitted; the resulting 3D printed hydrogel scaffold was designated sample S.
Comparative example 2
A 3D printed hydrogel scaffold was prepared according to the method of example 1, except that bone marrow mesenchymal stem cells were omitted; the resulting 3D printed hydrogel scaffold was designated sample B.
Comparative example 3
A 3D printed hydrogel scaffold was prepared according to the method of example 1, except that berberine was omitted; the resulting 3D printed hydrogel scaffold was designated sample C.
Test example 1
The compressive modulus of the 3D printed hydrogel scaffolds prepared in example 1 was tested using an Instron model 5944 100N capacity tester, wherein the compressive speed was 1mm/min, the experiment terminated at the breaking point of the strain stress curve, and the compressive young's modulus was calculated from the slope of the compressive strain stress curve in the linear region (10-20% strain).
For tissue engineering, compressive strength is one of the key factors for successful application of tissue engineering materials. Fig. 3 is a graph showing the compressive strength results of the 3D printed hydrogel scaffold prepared in example 1, and the results show that the 3D printed hydrogel scaffold prepared in example 1 exhibits good compressive strength, and the compressive young's modulus calculated by the compressive strain slope (10 to 20%) is 70KPa.
Test example 2
Vacuum freeze-drying the 3D printing hydrogel bracket prepared in the embodiment 1 for 24 hours to obtain a freeze-dried bracket, weighing the freeze-dried bracket and marking the freeze-dried bracket as MO, then putting the freeze-dried bracket into deionized water, weighing the freeze-dried bracket at different time points (10 min, 20min, 30min, 40min, 50min, 70min, 100min, 150min, 200min and 250 min), and sucking the deionized water on the surface of the bracket with filter paper to dry before each weighing, wherein the obtained quality is marked as MT, and stopping the experiment until the weight of the bracket has no obvious change; calculate the Swelling Ratio (SR) of the 3D printed hydrogel scaffold: sr= (MT-MO)/mo×100%. As a scaffold for tissue engineering, swelling of hydrogels helps to provide nutrition and promote metabolism during cell culture. The internal pore structure of the 3D printed hydrogel scaffold has a large impact on its swelling process. Fig. 4 is a graph showing the swelling ratio results of the 3D printed hydrogel scaffold prepared in example 1, which shows that the mass of the lyophilized scaffold immersed in water increases with time, and the mass of the scaffold after 70min tends to be smooth, 10 times the mass of the scaffold at the beginning.
And observing the microstructure of the bracket by adopting a scanning electron microscope, specifically, carrying out vacuum freeze drying on the 3D printing hydrogel bracket prepared in the embodiment 1 and the comparative examples 1-3 for 24 hours to obtain a freeze-dried bracket, plating gold on the surface of the freeze-dried bracket, standing for 1min, and then observing the microstructure on the surface and the inside of the freeze-dried bracket by using a Hitachi S-3400N electron microscope to obtain an SEM micrograph of the freeze-dried bracket. Fig. 5 is an SEM image of the freeze-dried scaffolds obtained after freeze-drying of the 3D printed hydrogel scaffolds prepared in example 1 and comparative examples 1-3, wherein S corresponds to the comparative example 1, b corresponds to the comparative example 2, c corresponds to the comparative example 3, bc corresponds to the example 1. As can be seen from fig. 5, the hydrogel scaffold with different components has similar surface morphology and internal structure, the hydrogel surface is smooth, and the internal structure has a plurality of pores; the presence of pores has many benefits for cells in the scaffold, the results are the same as those of the swelling ratio experiment, and the higher the porosity, the higher the swelling ratio.
Test example 3
The 3D printing hydrogel scaffold prepared in example 1 was soaked in PBS buffer solution with a concentration of 0.01M at room temperature, and the volume ratio of the 3D printing hydrogel scaffold to the PBS buffer solution is 1:20, a step of; the mass of the 3D printing hydrogel stent before soaking is marked as M1, and the mass of the stent obtained after soaking for different times is marked as M2; the test period time totaled 28 days. The 3D printed hydrogel scaffold Degradation Rate (DRT) was calculated as follows: drt=m1-M2/m1×100%.
Reasonable degradation rates are critical to tissue engineering scaffolds. If the degradation is too rapid, the scaffold will lose structural support to the cells; conversely, if the degradation rate is too slow, the appearance of new tissue is hindered. Fig. 6 is a graph showing degradation rate results of the 3D printing hydrogel scaffold prepared in example 1, and the results show that the degradation rate of the 3D printing hydrogel scaffold prepared in example 1 is stable, and the scaffold mass is 54% of the initial mass of the 3D printing hydrogel scaffold at 28 days.
Test example 4
The biocompatibility of the 3D printed hydrogel scaffolds prepared in example 1 and comparative example 3 was examined using a live/dead staining method, specifically, the cell viability of the 3D printed hydrogel scaffold-coated bone marrow mesenchymal stem cells (BMSCs) prepared in example 1 and comparative example 3 after 3 and 7 days of culture was evaluated by operating according to the procedure noted in the live/dead staining kit (Invitrogen) instruction, and the staining results (green as live cells, red as dead cells) were observed using a confocal laser microscope.
As a 3D printed biological scaffold, it is of paramount importance that the cells within the scaffold survive well. FIG. 7 is a graph of in vitro biocompatibility test results of 3D printed hydrogel scaffolds prepared in example 1 and comparative example 3, wherein C corresponds to comparative example 3 and BC corresponds to example 1. As can be seen from fig. 7, the live/dead staining was performed on the 3 rd and 7 th days in the culture solution, and the results showed that the viability of BMSCs cells in each group was 90% or more at the 3 rd day of culture, and there was no statistical difference; the growth effect of the cells is still good and the survival rate is more than 90% when the cells are cultured for 7 days.
Test example 5
The drug release rate of the 3D printed hydrogel scaffold prepared in example 1 was measured as follows: A3D printing hydrogel scaffold was prepared according to the method of example 1, wherein the volume of 3D bioprinting ink used was 1mL, the 3D printing hydrogel scaffold was soaked in 5mL of PBS buffer, PBS buffer soaked outside the scaffold was removed at time points of 2h, 8h, 24h, 2D, 4D, 8D, 10D, 12D, 14D, 16D, fresh PBS buffer was replaced, and the concentration of berberine in the removed PBS buffer was detected. The concentration of berberine is measured by ultraviolet spectrophotometry, and the detection wavelength is 345nm. The calculation formula is as follows: a=0.015744c+0.192 (a is absorbance value and C is berberine concentration). The plates used for the test were stored at 37 DEG C、5％CO 2 The rotation speed in the incubator is 60rpm; and calculating the release ratio of the berberine in the hydrogel according to the measured berberine concentration.
The high water content of hydrogels, the adjustable physical and mechanical properties, and a variety of synthetic strategies make them ideal drug delivery reservoirs. FIG. 8 is a release profile of berberine in the 3D printing hydrogel scaffold prepared in example 1, and the berberine in the hydrogel is burst released in the first 4 days, and the release rate reaches 72%; the berberine is released continuously after 12 days (5 th to 16 th days), but the release speed is slowed down, and the release curve reaches a stable level; on day 16, the berberine release rate was 89%.
Test example 6
1. Evaluation of in vitro osteogenic Capacity
The test example evaluates BMSCs in 3D printed hydrogel scaffolds for in vitro osteogenesis by detecting alkaline phosphatase activity, particularly in osteogenesis induction medium (containing 10 -7 The 3D printed hydrogel scaffolds prepared in example 1 and comparative example 3 were cultured in an aMEM basal medium of mol/L dexamethasone, 10mM sodium beta-glycerophosphate and 50 μg/mL ascorbic acid), osteogenic differentiation of BMSCs coated with the 3D printed hydrogel scaffold was induced, osteogenic induction medium was changed every 2 to 3 days, ALP (osteogenic differentiation markers) activity of BMSCs coated with 3D printed hydrogel scaffold was measured on days 7 and 14, respectively, the 3D printed hydrogel scaffold was washed three times with PBS buffer before detection, then lysate was added, and incubation was performed for 1h at 4 ℃; ALP activity was measured according to the procedure on the kit, absorbance was read at 405nm using a microplate, and the results were normalized to the total intracellular protein content measured by the BCA (bicinchoninicacid) protein assay kit, and expressed in nanomoles of pNP (nmol/min/mgprotein) produced per minute per milligram of protein (nmol/min/mgprotein).
FIG. 9 is a graph of ALP activity test results of BMSCs in 3D printed hydrogel scaffolds prepared in example 1 and comparative example 3 after 7 and 14 days of osteogenesis induction, wherein C corresponds to comparative example 3 and BC corresponds to example 1; * P is less than 0.05. As can be seen from fig. 9, there was no significant difference in ALP activity of BMSCs in the 3D printed hydrogel scaffolds with or without berberine at day 7; however, on day 14, the berberine-added 3D printed hydrogel scaffold of example 1 had higher ALP activity than the berberine-free 3D printed hydrogel scaffold of comparative example 3.
2. In vivo osteogenic evaluation
(1) Establishment of in vivo skull defect animal model
The experiment adopts SD rats (6 weeks) to establish a skull defect model, specifically, the rats are randomly divided into 5 groups of 5 rats each; removing hair on the epidermis of the rat skull, and separating the skull bone sticking membrane; preparing a bone defect on a rat skull by using a round drill hole with the diameter of 5mm, then implanting the 3D printing hydrogel scaffolds prepared in example 1 and comparative examples 1 to 3 into the bone defect, and setting a group of blank control groups; penicillin (8 ten thousand units/kg) was injected intramuscularly 3 days after the operation, rats were sacrificed at weeks 4 and 8, skull tissue was removed and fixed with 4% glutaraldehyde.
(2) Micro CT observation of bone formation
The separated rat skull sample is scanned by using the muCT 50 of SCANCO company, and the scanning parameters are as follows: the voltage is 70Kvp, the current is 100uA, the exposure time is 300ms, and a copper filter screen is adopted; and a three-dimensional image analysis of the skull and a quantitative analysis of the volume of new bone were performed by Invivo5.0 software.
Fig. 10 is a Micro CT 3D image (4 weeks and 8 weeks) of rat skull after implantation of 3D printed hydrogel scaffolds prepared in example 1 and comparative examples 1-3, wherein Con corresponds to the blank control group, S corresponds to the example 1, b corresponds to the example 2, c corresponds to the example 3, bc corresponds to the example 1. As can be seen from fig. 10, the bone formation effect of BC group was best at week 4, and the bone mass of C group and B group was slightly increased compared to Con group and S group; the osteogenic effect of BC group was still best compared to the other groups at week 8, with the increase in bone mass of C and B groups being more pronounced compared to Con and S groups.
FIG. 11 is a graph showing the measurement results of the formation volume of new bone at the skull defect site of rats after implantation of the 3D-printed hydrogel scaffolds prepared in example 1 and comparative examples 1 to 3 (for 4 weeks and 8 weeks, bone mass measurement was performed using Invivo5.0 software), wherein Con corresponds to the blank control group, S corresponds to the comparative example 1, B corresponds to the comparative example 2, C corresponds to the comparative example 3, BC corresponds to the comparative example 1; * P < 0.05 is compared with group B and group C, and #P < 0.01 is compared with group Con and group S. As can be seen from fig. 11, the formation of new bone was quantitatively expressed in terms of solid volume (BV) in the defect area, and the solid volume was superior for BC group at both weeks 4 and 8 to other groups.
(3) Histological analysis
After the microscopic CT detection is completed, 10% EDTA is used as decalcification liquid to soak and decalcifize the skull tissue, the decalcification liquid is replaced every 5 days, hematoxylin and eosin staining is carried out on the decalcified skull tissue after 2 months, and the skull tissue structure is observed; meanwhile, 1 rat was randomly selected from each group of sacrificed rats, and tissue sections were prepared by taking viscera, and HE staining was performed.
Fig. 12 is a graph of rat skull HE staining (4 weeks and 8 weeks) after implantation of the 3D printed hydrogel scaffolds prepared in example 1 and comparative examples 1-3, wherein Con corresponds to the blank control group, S corresponds to the example 1, b corresponds to the example 2, c corresponds to the comparative example 3, bc corresponds to the example 1. As can be seen from fig. 12, at weeks 4 and 8, bone defects of Con group were not produced with bone-like tissue, and only a small amount of fibrous hoof tissue was formed, consistent with the results obtained by Micro-CT observation; the best osteogenic effect of the BC group, the shortest distance between the two broken ends of the skull, shows that the BC group has more new bone tissue than other groups, and a great amount of new bone tissue is generated at the broken ends.
FIG. 13 is a plot of the HE staining of visceral tissue (x 100) of rats after implantation of the 3D printed hydrogel scaffolds prepared in example 1 and comparative examples 1-3, wherein Con corresponds to the blank control group, S corresponds to the example 1, B corresponds to the example 2, C corresponds to the example 3, and BC corresponds to the example 1. As can be seen from fig. 13, the visceral tissue structure of each group of rats was normal and unchanged. The material used for the 3D printing hydrogel scaffold provided by the invention is nontoxic to mammals, and has good biocompatibility in animal bodies.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. A biological ink comprises agarose, alginate, carboxymethyl chitosan, hydroxyapatite, berberine, bone marrow mesenchymal stem cells and PBS buffer solution; the concentration of agarose in the biological ink is 0.005-0.025 g/mL, the concentration of alginate is 0.03-0.07 g/mL, the concentration of carboxymethyl chitosan is 0.03-0.07 g/mL, the concentration of hydroxyapatite is 0.003-0.007 g/mL, the concentration of berberine is 0.005-0.02 g/mL, and the concentration of bone marrow mesenchymal stem cells is 1X 10 7 ～5×10 7 Individual cells/mL.
2. The bio-ink of claim 1 wherein the pH of the PBS buffer solution is 7.4.
3. A method of preparing the bio-ink according to claim 1 or 2, comprising the steps of:
dissolving agarose in PBS buffer solution to obtain a first mixture;
mixing the first mixture with an alginate to obtain a second mixture;
mixing the second mixture, carboxymethyl chitosan and hydroxyapatite to obtain a third mixture;
and mixing the third mixture, berberine and bone marrow mesenchymal stem cells to obtain the biological ink.
4. The preparation method of the 3D printing hydrogel bracket comprises the following steps:
3D bio-printing is carried out on the bio-ink according to claim 1 or 2 or the bio-ink prepared by the preparation method according to claim 3, so as to obtain a 3D printing bracket;
immersing the 3D printing support in soluble calcium salt water solution for crosslinking treatment to obtain the 3D printing hydrogel support.
5. The method according to claim 4, wherein the nozzle for 3D bioprinting has a diameter of 100 to 1000. Mu.m.
6. The method according to claim 4, wherein the printing temperature of the 3D bioprinting is 37 ℃ and the cooling plate is precooled to 4-10 ℃.
7. The method of claim 4, wherein the printing parameters of the 3D bioprinting include: each layer has a thickness of 0.05-0.5 mm, a layer number of 5-20 layers, a line spacing of 0.2-2 mm and a printing speed of 5-20 mm/s.
8. The process according to claim 4, wherein Ca in the aqueous solution of soluble calcium salt 2+ The concentration of (2) is 0.01-0.03 g/mL; the temperature of the crosslinking treatment is 25-37 ℃ and the time is 15-20 min.
9. The 3D printing hydrogel scaffold prepared by the preparation method of any one of claims 4 to 8, which comprises a hydrogel matrix, and hydroxyapatite, berberine and bone marrow mesenchymal stem cells loaded in the hydrogel matrix, wherein the hydrogel matrix is prepared from agarose, alginate and carboxymethyl chitosan in Ca 2+ Crosslinking is formed under the action of the catalyst.
10. Use of the 3D printed hydrogel scaffold of claim 9 for the preparation of bone repair regeneration material.
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