CN111330086A - Bionic artificial bone scaffold material and preparation method thereof - Google Patents

Bionic artificial bone scaffold material and preparation method thereof Download PDF

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CN111330086A
CN111330086A CN202010145880.7A CN202010145880A CN111330086A CN 111330086 A CN111330086 A CN 111330086A CN 202010145880 A CN202010145880 A CN 202010145880A CN 111330086 A CN111330086 A CN 111330086A
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artificial bone
bone scaffold
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scaffold material
tricalcium phosphate
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牛维
顾一帆
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Guangdong Hospital of Traditional Chinese Medicine
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Abstract

The invention relates to a bionic artificial bone scaffold material which is prepared by the following steps of (1) taking calcium carbonate, adding calcium hydrogen phosphate dihydrate 2 times according to a molar ratio, carrying out wet grinding for 2 hours by taking ethanol as a medium, carrying out hot air drying, then placing in a muffle furnace, gradually heating to 1000 ℃ to prepare β -tricalcium phosphate, and (2) taking β -tricalcium phosphate prepared in the step (1), adding magnesium oxide according to a mass ratio of β -tricalcium phosphate to magnesium oxide of 99: 1, carrying out wet grinding by taking ethanol as a medium, drying, carrying out dry grinding, and passing through a test sieve with the aperture of 100 microns to obtain the bionic artificial bone scaffold material.

Description

Bionic artificial bone scaffold material and preparation method thereof
Technical Field
The present invention relates to prosthetic materials, and in particular to phosphorus-containing inorganic materials characterized by their functional or physical properties, which can be 3D printed into prostheses.
Background
However, the defects of the allograft bone mainly include (1) immunological rejection, (2) low biological activity and mechanical property, (3) pathogen transmission risk, (3) calcium phosphate material has been widely used and researched as an artificial bone replacement material commonly used for bone defect repair at present, β -tricalcium phosphate has been proved to have good biological compatibility with natural bone mineral components, 95% of calcium phosphate can be degraded and absorbed by organisms after implantation, β -tricalcium phosphate has more advantages in bone reconstruction cycle fitting, and calcium phosphate material has been approved by the FDA as a calcium phosphate material commonly used in clinical FDA is generally in the form of osteogenic powder or calcium phosphate powder, is commonly used for bone defect repair, and can not be used for bone defect repair of bone defect of calcium phosphate alone, calcium phosphate is used as a calcium phosphate implant material for bone defect induction and bone defect induction of calcium phosphate regeneration of calcium phosphate of.
The patent application of the invention of 3D printing degradable β -tricalcium phosphate porous biological ceramic stent and a preparation method and application thereof (publication number: CN110092653A) is disclosed by the patent application on 08.06.2019, the patent application adopts 3D printing degradable β -tricalcium phosphate porous biological ceramic stent, the patent application of the invention of 04.19.2019 authorizes and announces a method for preparing a calcium phosphate porous biological ceramic stent based on a free extrusion type 3D printing technology and a preparation method thereof (publication number: CN109650872A), the patent discloses a method for preparing a calcium phosphate porous stent by using extrusion type 3D printing, the patent application of the invention of 03.16.2018 authorizes and announces a method for preparing a 3D printing stent as a bone repair biological material and a preparation method thereof (publication number: CN107802884A) by using native copper and a 2-tricalcium phosphate composite material, the patent application of the invention of the mesoporous ceramic stent by using calcined copper and the preparation of the bone repair biological material 3D printing, the invention of the mesoporous ceramic stent and the preparation method of the invention of the mesoporous ceramic stent by using the modified biological material prepared by using the calcined copper and the mesoporous ceramic stent prepared by the preparation technology of the mesoporous ceramic stent are disclosed by the patent application of the invention of the mesoporous calcium phosphate porous biological material, the invention of the mesoporous ceramic stent by using the patent application of.
Disclosure of Invention
In view of the defects of the prior art, the technical problem to be solved by the invention is a bionic artificial bone scaffold material, and a bone scaffold prepared from the material has excellent osteogenic differentiation and vascularization inducing performance.
The technical scheme for solving the technical problems is as follows:
a bionic artificial bone scaffold material is prepared by the following steps:
(1) adding calcium carbonate into calcium hydrogen phosphate dihydrate 2 times of the molar ratio, wet-milling with ethanol as medium for 2 hr, hot air drying, and gradually heating to 1000 deg.C in muffle furnace to obtain β -tricalcium phosphate (β -TCP);
(2) and (2) taking β -tricalcium phosphate prepared in the step (1), adding magnesium oxide according to the mass ratio of β -tricalcium phosphate to magnesium oxide of 99 to 1, wet-grinding by taking ethanol as a medium, drying, dry-grinding, and sieving by using a sieve with the pore diameter of 100 micrometers to obtain the bionic artificial bone scaffold material.
In the above scheme, the procedure of gradual heating is shown in the following table:
TABLE 1 muffle furnace time and temperature setting table
Figure BDA0002400706710000021
The bionic artificial bone scaffold material provided by the invention has excellent induced osteogenic differentiation and vascularization performances and can be used for preparing a bionic artificial bone scaffold, and the specific preparation method of the bone scaffold comprises the following steps:
(A) adding gelatin solution with the mass percentage concentration of 20% into the bionic artificial bone scaffold material, and enabling the concentration of the bionic artificial bone scaffold material in the gelatin solution to be 2g/mL to prepare 3D printing ink;
(B) filling the 3D printing ink prepared in the step (A) into a 3D printer and setting printing parameters;
(C) importing a three-dimensional reconstruction model of a femoral head necrosis patient into three-dimensional drawing software (such as common UG or proe) to draw a graph of the three-dimensional reconstruction model and store the graph as an STL file, importing the file into Slic3r to be converted into a geocode file and sending the geocode file to a 3D printer, and printing out a prosthesis model of the femoral head;
(D) freeze-drying the obtained prosthesis model, and sintering in a muffle furnace at 1250 ℃ for 2 hours; cooling, vacuum packaging, and placing60And (5) performing irradiation sterilization on the bionic artificial bone scaffold with a dose of 20kGy under Co gamma-ray to obtain the bionic artificial bone scaffold.
The bionic artificial bone scaffold material is prepared by specifically selecting β -tricalcium phosphate (β -TCP) and magnesium oxide in a ratio of 99: 1, so that the compressive strength of the scaffold is remarkably improved, the ALP activity and the osteogenic gene expression level in mesenchymal stem cells and the expression level of a vascularized gene in endothelial cells can be remarkably improved, the cell proliferation is remarkable, and the cytotoxicity is reduced.
Drawings
Fig. 1 is a three-dimensional reconstructed model of a necrotic femoral head.
Fig. 2 is a three-dimensional graph drawn according to the reconstructed model shown in fig. 1.
Detailed Description
Example 1 (preparation of scaffold Material)
In this example, the specific preparation method of the biomimetic artificial bone scaffold material is as follows:
(1) 28g (0.4mol) of calcium carbonate, 96.32g (0.8mol) of calcium hydrophosphate dihydrate and 185.5mL of anhydrous ethanol are put into a ball mill for wet milling for 2 hours, then are dried in a hot air furnace for 48 hours, and then are put into a muffle furnace to be gradually heated to 1000 ℃ according to the temperature rising program set in the following table 1 to prepare β -tricalcium phosphate;
TABLE 1 muffle furnace time and temperature setting table
Figure BDA0002400706710000031
(2) And (2) adding 59.4g of β -tricalcium phosphate prepared in the step (1) into 0.6g of magnesium oxide, putting the mixture into a planetary ball mill, adding absolute ethyl alcohol, wet-milling for 2 hours, taking out, drying, putting the mixture into a ball mill, and dry-milling for 2 hours, wherein the ball diameter is 100 microns, so as to obtain the bionic artificial bone scaffold material (hereinafter referred to as Mg-TCP), wherein the mixing ratio of the wet-milled material balls is Mg-TCP to the grinding balls, namely the absolute ethyl alcohol is 1: 2: 1.5, the wet-milling process parameter is the mass ratio of large grinding balls with the diameter of 1cm to small grinding balls with the diameter of 0.4cm being 1: 2, the grinding rotation speed is 300rpm, the mixing ratio of the dry-milled material balls is Mg-TCP to small grinding balls with the diameter of 0.4cm being 1: 2, and the dry-milling process parameter is the mixing ratio of large grinding balls with the diameter of 1cm to small grinding balls with the diameter of 0.4cm being 400 cm being 1: 2.
Example 2 preparation of bone scaffold
In this example, the specific preparation method of the bionic artificial bone scaffold is as follows:
(A) taking 4g of the bionic artificial bone scaffold material prepared in the example 1, and adding 2mL of gelatin solution with the mass percentage concentration of 20% to prepare 3D printing ink; wherein the gelatin solution with the mass percentage concentration of 20% is prepared by mixing gelatin purchased from Sigma-Aldrich company in the United states and medical water;
(B) loading the 3D printing ink prepared in the step (A) into a LivprintNorm3D printer of Guangzhou Meep regenerative medicine science and technology Limited and setting printing parameters; wherein the printing parameters are set to: selecting a dispensing needle head with the inner diameter of 420 mu m (22G), and setting the filling rate of the model to be 30%; the temperature of the needle head is 40 ℃, the temperature of the material cylinder is 50 ℃, the temperature of the material placing table is-5 ℃, the extrusion air pressure is 200Kpa, and the maximum moving speed of a printer coordinate system is 30[ pulse/ms ];
(C) importing a three-dimensional reconstruction model (shown in figure 1) of the femoral head of a patient with femoral head necrosis into proe three-dimensional drawing software to draw a complete femoral head three-dimensional graph (shown in figure 2) of the patient and store the graph as an STL file, importing the file into Slic3r to be converted into a geocode file and sending the gcode file to a 3D printer, and printing out a prosthesis model of the femoral head of the patient;
(D) freeze-drying the obtained prosthesis model, and sintering in a muffle furnace at 1250 ℃ for 2 hours; cooling, vacuum packaging, and placing60And (5) performing irradiation sterilization on the bionic artificial bone scaffold with a dose of 20kGy under Co gamma-ray to obtain the bionic artificial bone scaffold.
Example 3 (Effect testing experiment)
Sample (1 Mg-TCP): example 2 the prepared biomimetic artificial bone scaffold;
control 1(0 Mg-TCP): modifying the addition of magnesium oxide to 0, firstly preparing the bionic artificial bone scaffold material according to the embodiment 1, and then preparing the bionic artificial bone scaffold by using the scaffold material according to the method of the embodiment 2;
control 2(2 Mg-TCP): modifying the addition of magnesium oxide to 2, firstly preparing the bionic artificial bone scaffold material according to the embodiment 1, and then preparing the bionic artificial bone scaffold by using the scaffold material according to the method of the embodiment 2;
control 3(4 Mg-TCP): the addition of magnesium oxide was modified to 4, and the biomimetic artificial bone scaffold material was prepared according to example 1, and then the scaffold material was used to prepare the biomimetic artificial bone scaffold according to the method of example 2.
1. Experimental methods
1.1 evaluation of physical and chemical Properties
The experiment detects and characterizes the physical and chemical properties, phase composition, microstructure, porosity and the like of the prepared novel 3D printing magnesium ion doped tricalcium phosphate bionic artificial bone scaffold by adopting the following methods. The methods and instrumentation used for the experimental tests were as follows:
1.1.1 topography Observation
The pore morphology, internal pore connectivity, surface crystallization of the scaffolds were observed by a field emission scanning electron microscope (SEM; ULTRA 55, LEO Gemini, Zeiss, Germany) equipped with an energy dispersive X-ray spectrometer (EDS; XFlash 6130, Bruker, Germany) with an acceleration voltage set at 15 kV. When microscopic morphology of the stent is observed by using SEM, the micro-area of the stent fiber is simultaneously analyzed for element types, distribution and content by using the attached EDS. All samples were mounted on aluminum stubs by conductive glue and coated with a thin gold layer prior to SEM observation. And directly outputting and storing data acquired by the SEM and the EDS in a digital format image.
1.1.2 porosity
Apparent porosity of the scaffold, the actual volume of the scaffold (marked V) was determined by hydrostatic weighing using a pycnometer based on archimedes' principle1) The calculation equation of (a) is:
V1＝(W0+W1-W2)/ρwater
where ρ iswaterIs the density of water at 25 ℃ (0.99705 g/cm)3)，W0(g) Is the mass of a pycnometer filled with ultrapure water, W1(g) Is the mass of each rack weighed. The holder is dipped into a pycnometer and the inside of the holder is filled with ultrapure water by vacuum, after which the pycnometer is filled, weighed and marked W2(g) In that respect The equation for the apparent porosity of the scaffold is calculated as:
porosity (%) ═ (V)0-V1)/V0×100％
Wherein V0(cm3) Is the apparent volume of the scaffold, calculated from the length, width and height of the scaffold.
1.1.3 mechanical Properties
The compressive strength of the stent was tested using a universal material tester (E34, tested, MTS, USA). The stent sample to be tested was a 10mm × 10mm × 8mm rectangular solid, the upper and lower surfaces of the stent were polished with sandpaper before the test started, and the compression test rate was set to 0.5 mm/min.
1.1.4 in vitro ion Release
After the scaffold mass was accurately weighed, the scaffold was soaked in complete medium at a ratio of 0.1g/mL, shaken at 37 ℃ with 60r/min using a shaker to simulate in vivo degradation, and the soak solutions were collected at 1, 3, 5, 7, 9, 11, and 13 days, respectively, and supplemented with fresh equal amount of complete medium. The concentration of magnesium ions released in the collected leachate was measured using inductively coupled plasma atomic emission spectrometry (ICP-AES; Perkinelmer, Optimal 5300DV, USA).
1.2 cell experiments
1.2.1 cell proliferation assay
A48-well cell culture plate was prepared and 500. mu.L of cell suspension (cell density 1 x 10) was added to each well4/mL), transferred into 5% CO at 37 deg.C2Culturing in a cell culture box with saturated humidity; after 24 hours, removing the culture medium, adding a support real-time leaching solution, and changing the solution every other day; detection time points were set at 1, 4, and 7 days after induction culture, and 4 replicates per group of material were set at each time point. Proliferation of hBMSCs and HUVECs was measured at each assay time point using Cell Counting Kit-8 (CCK-8): carefully sucking up the composite scaffold leach liquor in each well, and adding 250 μ L of CCK-8 working solution (basal medium + 10% CCK8 stock solution) to each well; incubation at 37 ℃ for 1 hour; taking out the generated orange reaction solution according to 100 mu L/hole, transferring the orange reaction solution into a 96-hole plate, eliminating air bubbles in the hole plate, and reading the OD value at 450nm by using an enzyme label; and (4) converting the measured OD value into the number of the cells according to a self-made cell-OD value standard curve and carrying out statistical analysis and calculation.
1.2.2 quantitative determination of alkaline phosphatase Activity
48 well cell culture plates 500. mu.L of hBMSCs cell suspension (cell density 1 x 10) was added per well5/mL), was charged with 5% CO at 37 ℃2Culturing in a cell culture box with saturated humidity, attaching most cells to the wall after 24 hours, replacing culture medium with bracket real-time leaching liquor (containing 10mM β -glycerophosphate, 10nM dexamethasone and 50mg/mL L-ascorbic acid) for induced culture, performing liquid replacement operation every other day, setting 7, 10 and 14 days after cell planting as detection time points, and setting 6 parallel samples (4 quantitative detections +2 definite detections) for each group of materials at each time pointSexual staining).
And (2) quantitatively detecting the ALP activity in the hBMSCs cell lysate by using an alkaline phosphatase activity detection kit at each time point, reading an OD value with the wavelength of 405nm by using a microplate reader, and quantitatively calculating the ALP activity by using an ALP activity standardized calculation formula: ALP activity (unit/μ g protein) ═ a/V/T/P, where a is the amount of finally produced pNP in the sample (μmol, calculated from OD value conversion by preparing a standard curve), V is the volume per repeated sample (ml) of each group, T is the reaction time (min), and P is the total protein content per cell of each group (μ g).
1.2.3 expression detection of osteogenesis and angiogenesis-related genes
Detecting osteogenesis related genes (Runx-2, Col-I, OCN, ALP and BSP) expressed by hBMSCs and vascularization related genes (VEGF and eNOS) expressed by HUVECs by RT-qPCR; GAPDF is selected as housekeeping gene internal reference.
Extracting and purifying total RNA in hBMSCs and HUVECs cells by using a double-column centrifugation method: add 350 μ L of Buffer RL to the 6-well culture plate, pipette 10 times to break gDNA; after loading gDNA Filter MiniColumn in a 2ml enzyme free EP tube, the cell lysate was transferred to a Filter column and centrifuged at high speed: 14000G, 2 minutes; discarding the gDNA filter column, adding 70% ethanol (prepared by absolute ethanol + DEPC treated water) with equal volume to the filtrate, and sucking with a pipette 5 times; HiPure RNA Mini Column was placed in a 2ml enzyme-free EP tube, 70% ethanol-cell lysis mixture was transferred to a spin Column and centrifuged at 12000G for 1 min; discard the filtrate, put the column back into the EP tube, add 600. mu.l of BufferRW1 to the column, centrifuge again at 12000G for 1 min; discarding the filtrate, loading the spin column back into EP tube, adding 600 μ L Buffer RW2, 12000G, centrifuging for 1 min, repeating for 2 times; the column was returned to the EP tube and centrifuged at 12000G for 2 minutes to clear all filtrate; using a new 1.5ml EP tube, load into the column and add 30. mu.L RNase Free Water to the center of the membrane in the column; standing at room temperature for 2 minutes, and centrifuging at 12000G for 1 minute; the spin column was discarded, and the filtrate in the EP tube was the total RNA extracted from the cells.
Quantitatively detecting the extracted total RNA of the cells by using Nanodrop and storing data; mu.L gDNA EraserBuffer + 1. mu.g total RNA was added to a 0.2mL EP tube, and DEPC-treated water was supplemented to bring the total reaction system to 10. mu.L; water bath at 37 ℃ for 2 minutes to remove gDNA interference; add 10. mu.L of 2 XMaster Mix (4. mu.L of 5X RT Buffer + 2. mu.L of 10X RT Primer Mix + 2. mu. L M-MLV reverse transcriptase + 2. mu.L of DEPC-treated water) per tube; transfer the EP tube to a qPCR instrument, set the reverse transcription reaction program: 60 minutes at 42 ℃ to 10 minutes at 80 ℃; after the reaction was complete, the EP tubes were quickly transferred to a low temperature ice box. Taking 2 mu L of synthesized cDNA to each tube of the PCR eight-connected tube, adding 3 mu M of forward and reverse primer sequences, 10 mu L of 2X SYBR Green qPCR Mix and 6 mu L of DEPC-treated water into each hole, and fully and uniformly mixing to obtain a 20 mu L PCR reaction system; transferring the eight-tube to a PCR instrument, and setting a reaction program: cDNA unwinding (10 min cDNA unwinding at 95 ℃) -40 amplification cycles (10 sec at 95 ℃ to 30 sec at 60 ℃) -a dissolution curve (15 sec at 95 ℃ to 60 sec at 95 ℃ to 15 sec at 95 ℃); after the PCR reaction is finished, deriving a dissolution curve and a Ct value, calculating the expression level of each gene according to a formula, and carrying out statistical analysis calculation:
Gene expression level＝2-ΔCt(ΔCt＝Cttarget gene-CtGAPDH)
2. results of the experiment
2.1 evaluation of physical and chemical Properties
2.1.1 topography Observation
Under the low power SEM, micropores on the upper surface and the lower surface of the scaffold are approximately square, the pore diameter is about 400 microns, micropores on the side surface are approximately rectangular, the size is 300 microns × 100 microns, and the diameter of a scaffold fiber line is about 450 microns.
2.1.2 porosity of scaffolds
The porosity of the scaffold is between 61.23% and 65.02%, and can be matched with the porosity (50% to 90%) of natural cancellous bone.
Table 2 apparent porosity of scaffolds
Figure BDA0002400706710000071
Figure BDA0002400706710000072
2.1.3 mechanical Property analysis of stents
The results of the maximum pressure bearing and Young modulus of the four groups of brackets are shown in Table 2, the maximum pressure bearing of all the brackets is more than 3MPa, and the mechanical property standard (3-30MPa) of human cancellous bone can be achieved.
TABLE 3 compressive Strength of scaffolds
Figure BDA0002400706710000073
Figure BDA0002400706710000074
2.1.4 in vitro ion Release detection of scaffolds
Table 4 shows the change in magnesium ion concentration in Mg-TCP scaffold leach liquor over 14 days.
TABLE 4 continuous release of magnesium ions from scaffolds (mM)
Figure BDA0002400706710000075
2.2 cell experiments
2.2.1 cell proliferation
The results of the cell proliferation assay using CCK-8 are shown in Table 5.
TABLE 5 cell proliferation
Figure BDA0002400706710000076
Figure BDA0002400706710000077
2.2.2 quantitative determination of alkaline phosphatase Activity
The results of quantitative determination of ALP activity in hBMSCs cells cultured by four groups of Mg-TCP leach liquor induction are shown in Table 6.
TABLE 6 ALP quantitative determination results
Figure BDA0002400706710000078
Figure BDA0002400706710000079
2.2.3 detection of expression of osteogenesis and angiogenesis-related genes
The expression of the osteogenesis related genes of hBMSCs cultured in the leaching solution of the four Mg-TCP porous scaffolds at 7 days and 14 days of culture is shown in Table 7.
TABLE 7 osteogenesis-related Gene expression of hBMSCs (Rel.to GAPDH)
Figure BDA0002400706710000081
The expression of angiogenesis-related genes at 7 days and 14 days in HUVECs cultured in four Mg-TCP porous scaffold extracts is shown in Table 8.
TABLE 8 expression of vascularization-related genes of HUVECs (Rel. to GAPDH)
Figure BDA0002400706710000082
3. Conclusion of the experiment
The experimental results prove that the pore size, porosity and mechanical properties of the 3D printed magnesium ion doped tricalcium phosphate bionic artificial bone scaffold can be matched with those of natural cancellous bone, and the scaffold has better osteogenesis inducing performance and vascularization inducing performance by modifying magnesium oxide which is 0.01 time of the total mass of the doped material in β -tricalcium phosphate material.

Claims (2)

1. A bionic artificial bone scaffold material is prepared by the following steps:
(1) adding calcium carbonate into calcium hydrogen phosphate dihydrate 2 times of the molar ratio, wet-milling for 2 hours by using ethanol as a medium, drying by hot air, and then placing in a muffle furnace to gradually heat to 1000 ℃ to prepare β -tricalcium phosphate;
(2) and (2) taking β -tricalcium phosphate prepared in the step (1), adding magnesium oxide according to the mass ratio of β -tricalcium phosphate to magnesium oxide of 99 to 1, wet-grinding by taking ethanol as a medium, drying, dry-grinding, and sieving by using a sieve with the pore diameter of 100 micrometers to obtain the bionic artificial bone scaffold material.
2. A preparation method of a bionic artificial bone scaffold comprises the following steps:
(A) adding gelatin solution with the mass percentage concentration of 20% into the bionic artificial bone scaffold material of claim 1, and enabling the concentration of the bionic artificial bone scaffold material in the gelatin solution to be 2g/mL to prepare 3D printing ink;
(B) filling the 3D printing ink prepared in the step (A) into a 3D printer and setting printing parameters;
(C) importing a three-dimensional reconstruction model of a femoral head necrosis patient into three-dimensional drawing software to draw a graph of the three-dimensional reconstruction model and store the graph as an STL file, importing the file into Slic3r to be converted into a gcode file and sending the gcode file to a 3D printer, and printing a prosthesis model of the femoral head;
(D) freeze-drying the obtained prosthesis model, and sintering in a muffle furnace at 1250 ℃ for 2 hours; cooling, vacuum packaging, and placing60And (5) performing irradiation sterilization on the bionic artificial bone scaffold with a dose of 20kGy under Co gamma-ray to obtain the bionic artificial bone scaffold.
CN202010145880.7A 2020-03-05 2020-03-05 Bionic artificial bone scaffold material and preparation method thereof Pending CN111330086A (en)

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