CN110028840B - Preparation method of nano-cellulose biological printing gel ink - Google Patents
Preparation method of nano-cellulose biological printing gel ink Download PDFInfo
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- CN110028840B CN110028840B CN201910354045.1A CN201910354045A CN110028840B CN 110028840 B CN110028840 B CN 110028840B CN 201910354045 A CN201910354045 A CN 201910354045A CN 110028840 B CN110028840 B CN 110028840B
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Abstract
The invention discloses a preparation method of nano-cellulose biological printing gel ink, which comprises the steps of pretreating paper pulp fibers by adopting high-pressure sterilization and alkali/acid sequential extraction, and preparing ultrapure nano-cellulose hydrogel by using TEMPO catalytic oxidation combined with high-pressure homogenization treatment; biologically coupling the sterilized xyloglucan and a cell adhesion factor to prepare bioactive xyloglucan; and dissolving the bioactive xyloglucan in sterile water, adding the nano-cellulose hydrogel and the sterilized gel reinforcing agent, and uniformly mixing to obtain the ultrapure nano-cellulose bioprinting gel ink. The prepared ink has excellent biocompatibility, no cytotoxicity and biodegradability; the highly hydrated three-dimensional network structure can simulate and reduce a real extracellular matrix microenvironment and structure to the maximum extent, and provides an ideal microenvironment for cell adhesion, growth, reproduction and differentiation. The pyrogenic and allergenic impurities are far lower than the pharmacopoeia regulations and medical clinical threshold values, and the application is wide.
Description
Technical Field
The invention relates to a preparation method of gel ink, in particular to a preparation method of ultrapure nano-cellulose bioprinting gel ink, belonging to the technical field of new biological and medical material manufacturing.
Background
The nano-cellulose is easy to form hydrogel in the preparation process, and the highly hydrated three-dimensional network structure of the nano-cellulose can well simulate extracellular matrix in human organ tissues, so that a good 3D microenvironment is provided for cell adhesion, growth and propagation. Therefore, at present, the development and utilization of 3D printing cellulose-based materials at home and abroad mainly focus on the biomedical application fields of artificial organs, tissue engineering, wound repair and the like.
The home and abroad commercialized nano-cellulose-based biological printing ink mainly comprises Swedish nano-cellulose-sodium alginate composite ink
The ink is gel ink prepared by mixing nano-cellulose and sodium alginate according to a certain proportion. Although it is not limited to
The ink can be used for smoothly performing 3D printing on different biomedical structures, but the 3D printing based on the composite ink still has a series of biocompatibility defects and shortcomings in the application of new biomedical materials:
(1) potential heat-sensitizing impurities (endotoxin, (1,3) -beta-D-glucan) in the preparation process of the nano fiber and the sodium alginate cannot be strictly controlled according to related pharmacopoeia or medical clinical threshold;
(2) sodium alginate and nano-cellulose are both biologically inert, have no cell adhesion and cannot directly promote cell growth and propagation, so that the cell proliferation is slow;
(3) the mechanical strength of the printing structure is low or is not matched with the strength of natural tissues, and the strength of the printing structure is weakened along with the loss of cross-linked calcium ions along with the lapse of cell culture or in vivo transplantation time;
(4) due to the fact that cellulose is difficult to carry out effective biodegradation in a human body, the biodegradation rate of the printing support structure is not matched with that of new tissue formation;
(5) cells in a larger and thicker printed structure are difficult to exchange effective nutrient and metabolic wastes with the outside, so that the activity of the cells is reduced;
(6) the cross-linking agent (e.g., calcium ions) used during printing can cause damage to cell viability.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a preparation method of a biodegradable nano-cellulose biological printing gel ink with excellent biocompatibility, no cytotoxicity.
In order to achieve the above object, the present invention adopts the following technical solutions:
a preparation method of ultrapure nano-cellulose bioprinting gel ink comprises the following steps:
s1, pretreating paper pulp fibers by adopting high-pressure sterilization and alkali/acid sequential extraction, and then carrying out TEMPO catalytic oxidation combined high-pressure homogenization treatment to prepare the ultrapure nanocellulose hydrogel;
s2, biologically coupling the sterilized xyloglucan and a cell adhesion factor to prepare the bioactive xyloglucan;
and S3, dissolving the bioactive xyloglucan in sterile water, adding the nano-cellulose hydrogel and the sterilized gel reinforcing agent, and uniformly mixing to obtain the ultrapure nano-cellulose bioprinting gel ink.
The autoclaving and alkali/acid sequential extraction pretreatment in step S1 above, comprising the steps of:
a1, stirring and dispersing bleached paper pulp fibers in sterile water, adding sodium hydroxide and hydrogen peroxide, stirring, sterilizing at high pressure, and washing to be neutral;
and A2, stirring and dispersing in sterile water, adding hydrochloric acid, stirring, sterilizing at high pressure, and washing to be neutral.
Further, in the step A1, 100g of pulp fiber, 3L of sterile water, 5-10g of sodium hydroxide and 2-5g of hydrogen peroxide are sterilized for 15-60min;
in the step A2, 3L of sterile water and 10-30g of hydrochloric acid are used, and the sterilization time is 15-60min.
The TEMPO catalytic oxidation combined high-pressure homogenization treatment in the step S1 comprises the following steps:
b1, adding TEMPO, sodium bromide and sodium hypochlorite into the pretreated paper pulp fibers, adjusting and keeping the pH value to 10.0-10.5, and stirring for reaction until the pH value of the system is stable;
b2, filtering and washing with sterile water until the conductivity of the filtrate is lower than 5 mu S/cm;
and B3, dispersing the washed pulp fibers in sterile water, and homogenizing under high pressure at the pressure of 200-600bar and 800-1800bar respectively to prepare the ultrapure nanocellulose hydrogel.
Furthermore, the concentration of the nano-cellulose hydrogel is more than 3%, and the content of endotoxin is less than 5EU/g, (1,3) -beta-D-glucan is less than 10 mu g/g.
Further, TEMPO in the step B1 is 0.5-1g, sodium bromide is 2-6g, and the concentration of sodium hypochlorite is 15%, and the mass is 300-600g;
the amount of sterile water used in step B2 was 2L.
The sterilization of xyloglucan in the step S2 is as follows: dissolving xyloglucan in sterile water, and autoclaving;
the sterilization of the gel enhancer in step S3 is: dissolving the gel enhancer in sterile water, and autoclaving.
The bioconjugation in the step S2 comprises the following steps:
c1, precipitating the sterilized xyloglucan in ethanol, filtering, cleaning, and freeze-drying to obtain purified xyloglucan;
and C2, dissolving the purified xyloglucan in sterile water, adding a cell adhesion factor, stirring at room temperature for reaction, dialyzing by a dialysis bag and sterile running water, and freeze-drying to obtain the bioactive xyloglucan.
Further, the ethanol in the step C1 is 3L;
the sterile water in the step C2 is 1L, the cell adhesion factors are 1-3g, the reaction time is 6-24h, the dialysis bag is 3.5K, and the dialysis time is 12-48h.
The bioactive xyloglucan in the step S3 is 0.5-2g, 10-50ml of sterile water, 100g of nano-cellulose hydrogel and 0.02-1.0g of gel reinforcing agent.
The invention has the advantages that:
according to the preparation method of the ultrapure nano-cellulose bio-printing gel ink, the prepared bio-printing gel ink has excellent biocompatibility, no cytotoxicity and biodegradability; the highly hydrated three-dimensional network structure can simulate and reduce a real extracellular matrix microenvironment and structure to the maximum extent, and provides an ideal microenvironment for cell adhesion, growth, reproduction and differentiation. The raw materials are pretreated to remove pyrogenic and allergenic impurities (endotoxin, (1,3) -beta-D-glucan) in the raw materials, and the content of the impurities is ensured to be far lower than pharmacopeia regulations and medical clinical thresholds, so that the product can be applied to the fields of tissue engineering, biological scaffold construction, 3D cell culture, drug screening, 3D printing and the like.
The invention solves the problem that pyrogenic sensitization impurities in other nano-cellulose gel inks cannot be controlled, overcomes the defect of biological inertia of other nano-cellulose gel inks, overcomes the defect of insufficient mechanical properties of other biological inks, and has the defect of influencing cell activity due to calcium ion crosslinking, thereby having strong practicability and wide applicability.
Drawings
Figure 1 is a structural diagram of a biological stent for bioprinting using an ultra-pure nanocellulose gel ink prepared according to the present invention.
Detailed Description
The invention is described in detail below with reference to the figures and the embodiments.
A preparation method of ultrapure nano-cellulose bio-printing gel ink comprises the following steps:
(1) 100g of bleached pulp fiber is stirred and dispersed evenly in 3L of sterile water, 10g of sodium hydroxide and 2g of hydrogen peroxide are added, stirred evenly and autoclaved for 60 minutes;
dissolving xyloglucan 10g in 1L sterile water, and autoclaving for 30 min;
5g of gel enhancer was dissolved in 100 ml of sterile water and autoclaved for 20 minutes.
(2) Filtering and washing the pulp fiber in the step (1) by using sterile water until the pH is neutral.
(3) And (3) dispersing the pulp fibers cleaned in the step (2) in 3 liters of sterile water, adding 30 grams of hydrochloric acid, uniformly stirring, and carrying out autoclaving for 60 minutes.
(4) And (4) filtering and washing the pulp fibers in the step (3) by using sterile water until the pH is neutral.
(5) And (3) dispersing the paper pulp fibers cleaned in the step (4) in 5 liters of sterile water, stirring, and preparing the high-purity and high-concentration (the concentration is more than 3%) nanocellulose hydrogel by adopting TEMPO catalytic oxidation combined with high-pressure homogenization treatment.
(1 g TEMPO (2,2,6,6-tetramethylpiperidinyloxy or 2,2,6,6-tetramethylpiperidinyloxy), 5g sodium bromide, 500 g sodium hypochlorite at 15% concentration were added, the pH was adjusted and maintained to 10.5, the reaction was stirred at room temperature until the pH of the system was not changed, filtered and washed with sterile water until the conductivity of green leaves was less than 5. Mu.S/cm.
And (4) dispersing the pulp fibers cleaned in the step (4) in 2 liters of sterile water, uniformly stirring, and carrying out high-pressure homogenization at pressures of 500bar and 1500bar respectively to prepare the high-purity and high-concentration nano-cellulose hydrogel. )
(6) And (2) precipitating the xyloglucan solution sterilized in the step (1) into 3 liters of ethanol, filtering, washing, freezing and drying to obtain the purified xyloglucan.
(7) And (3) dissolving the purified xyloglucan obtained in the step (6) in 1 liter of sterile water, adding 3g of cell adhesion factor (RGD), stirring and reacting for 24 hours at room temperature, dialyzing for 24 hours in a 3.5K dialysis bag and sterile running water, and freeze-drying to obtain the bioactive xyloglucan.
(8) And (3) dissolving 1g of the bioactive xyloglucan obtained in the step (7) in 50ml of sterile water, adding 100g of the nano-cellulose hydrogel obtained in the step (5), adding 0.5 g of the gel enhancer solution obtained in the step (1), and uniformly mixing to obtain a finished product of the ultra-pure nano-cellulose bioprinting gel ink.
TABLE 1
Table 1 above shows the content of heat-sensitizing impurities in the ultrapure nanocellulose bioprinting gel ink prepared by the examples of the present invention, and the printing performance of the bioprinting bioscaffold using the ink.
As can be seen from Table 1, the endotoxin and (1,3) -beta-D-glucan content of the ink is well below the European pharmacopoeia and clinical thresholds.
As shown in fig. 1, a structure diagram of a biological scaffold printed by using the ultra-pure nano-cellulose bio-printing gel ink according to the embodiment of the present invention shows that the ink disclosed in the present invention can be used for bio-printing to smoothly print a biological scaffold structure, the printed structure has high resolution, and the printed structure does not need to be cross-linked and enhanced by using calcium ions.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It should be understood by those skilled in the art that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the scope of the present invention.
Claims (7)
1. A preparation method of nano-cellulose bio-printing gel ink is characterized by comprising the following steps: s1, adopting high-pressure sterilization and alkali/acid sequence extraction to pretreat pulp fibers, and then using TEMPO catalytic oxidation in combination with high-pressure homogenization to prepare nano-cellulose hydrogel with the concentration of 3 percent and the endotoxin content of less than 5EU/g (1,3) -beta-D-glucan content of less than 10 mu g/g;
the pretreatment comprises the following steps:
a1, stirring and dispersing bleached paper pulp fibers in sterile water, adding sodium hydroxide and hydrogen peroxide, stirring, sterilizing at high pressure, and washing to be neutral;
a2, stirring and dispersing in sterile water, adding hydrochloric acid, stirring, sterilizing at high pressure, and washing to be neutral; s2, biologically coupling the sterilized xyloglucan and a cell adhesion factor to prepare bioactive xyloglucan; s3, dissolving 0.5-2.0g of bioactive xyloglucan in 10-50ml of sterile water, adding 100g of nano-cellulose hydrogel and 0.02-1.0g of sterilized gel reinforcing agent, and uniformly mixing to obtain the nano-cellulose bioprinting gel ink.
2. The preparation method of the nano-cellulose bio-printing gel ink according to claim 1, characterized in that in the step A1, the pulp fiber is 100g, the sterile water is 3L, the sodium hydroxide is 5-10g, the hydrogen peroxide is 2-5g, and the sterilization time is 15-60min;
in the step A2, 3L of sterile water and 10-30g of hydrochloric acid are used, and the sterilization time is 15-60min.
3. The method for preparing a gel ink for nano-cellulose biological printing according to claim 1, wherein TEMPO catalytic oxidation in step S1 is combined with high-pressure homogenization treatment, and the method comprises the following steps:
b1, adding TEMPO, sodium bromide and sodium hypochlorite into the pretreated paper pulp fibers, adjusting and keeping the pH value to 10.0-10.5, and stirring for reaction until the pH value of the system is stable;
b2, filtering and washing with sterile water until the conductivity of the filtrate is lower than 5 mu S/cm;
and B3, dispersing the washed pulp fibers in sterile water, and homogenizing under high pressure at the pressure of 200-600bar and 800-1800bar respectively to prepare the nano-cellulose hydrogel.
4. The method for preparing a gel ink for nano-cellulose biological printing according to claim 3, wherein TEMPO in the step B1 is 0.5-1.0g, sodium bromide is 2-6g, and the concentration of sodium hypochlorite is 15% and the mass is 300-600g;
the amount of sterile water used in step B2 was 2L.
5. The method for preparing a nano-cellulose bio-printing gel ink according to claim 1, wherein the sterilization of xyloglucan in the step S2 is: dissolving xyloglucan in sterile water, and autoclaving;
the sterilization of the gel enhancer in step S3 is: dissolving the gel enhancer in sterile water, and autoclaving.
6. The method for preparing a nano-cellulose bio-printing gel ink according to claim 1, wherein the bio-coupling in the step S2 comprises the following steps:
c1, precipitating the sterilized xyloglucan in ethanol, filtering, cleaning, and freeze-drying to obtain purified xyloglucan;
and C2, dissolving the purified xyloglucan in sterile water, adding a cell adhesion factor, stirring at room temperature for reaction, dialyzing by a dialysis bag and sterile running water, and freeze-drying to obtain the bioactive xyloglucan.
7. The method for preparing a nano-cellulose bio-printing gel ink according to claim 6, wherein the ethanol in the step C1 is 3L;
the sterile water in the step C2 is 1L, the cell adhesion factors are 1-3g, the reaction time is 6-24h, the dialysis bag is 3.5K, and the dialysis time is 12-48h.
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| WO2013063602A1 (en) * | 2011-10-27 | 2013-05-02 | Bc Genesis Llc | Design and fabrication of biomimetic soft tissue scaffolds based on controlled assembly of highly crystalline beta-glucan nanofibrils and use in reconstructive surgery |
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| EP1961432A1 (en) * | 2007-02-26 | 2008-08-27 | Swetree Technologies Ab | Implantable material comprising cellulose and the glycopeptide xyloglucan-GRGDS |
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| JP2010518998A (en) * | 2007-02-26 | 2010-06-03 | スウェツリー・テクノロジーズ・アクチボラゲット | Implant materials for medical or surgical applications |
| WO2013063602A1 (en) * | 2011-10-27 | 2013-05-02 | Bc Genesis Llc | Design and fabrication of biomimetic soft tissue scaffolds based on controlled assembly of highly crystalline beta-glucan nanofibrils and use in reconstructive surgery |
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