CN108567993B - Method for constructing artificial intelligence pancreas for reducing blood sugar based on 3D printing - Google Patents

Method for constructing artificial intelligence pancreas for reducing blood sugar based on 3D printing Download PDF

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CN108567993B
CN108567993B CN201810236193.9A CN201810236193A CN108567993B CN 108567993 B CN108567993 B CN 108567993B CN 201810236193 A CN201810236193 A CN 201810236193A CN 108567993 B CN108567993 B CN 108567993B
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printing
pancreas
gelatin
blood glucose
insulin
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CN108567993A (en
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薛巍
阮淼亮
戴箭
宋镕光
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Jinan University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/222—Gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y80/00—Products made by additive manufacturing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/602—Type of release, e.g. controlled, sustained, slow

Abstract

The invention discloses a method for constructing an artificial intelligent pancreas for reducing blood sugar based on 3D printing, and relates to the field of 3D biological printing and artificial pancreas. The method comprises the steps of preparing acetalized dextran-loaded insulin nanoparticles, preparing hydrogel for printing, preparing hydrogel support bath, preparing 3D printing and the like. The artificial intelligent pancreas prepared by 3D printing has the advantages of good biocompatibility and biodegradability, and can control the release of insulin according to the real-time blood glucose concentration, so that the aims of intelligently reducing blood glucose and controlling the blood glucose concentration within a normal range for a long time are fulfilled, and multiple blood glucose detection and subcutaneous injection are avoided. The invention does not need to carry portable equipment, does not need to replace batteries, catheters and the like, can greatly reduce the burden of patients, and has more outstanding value of application basic research and market development prospect with great potential.

Description

Method for constructing artificial intelligence pancreas for reducing blood sugar based on 3D printing
Technical Field
The invention relates to the field of 3D biological printing and artificial pancreas, in particular to a method for constructing an artificial intelligent pancreas for reducing blood sugar based on 3D printing.
Background
At present, insulin is mainly injected subcutaneously for many times according to the measurement result by continuously detecting blood sugar, which brings much pain and inconvenience to patients. In addition, the injected insulin has the defects of unstable absorption, peak concentration, incapability of well simulating insulin secretion, difficulty in accurately calculating the injection amount according to the absorption level of a patient, possibility of causing hypoglycemia due to calculation errors and the like. Administration of insulin is required to provide a precise dose of insulin at the appropriate time to restore blood glucose concentration to normal levels. Because of this property, researchers have devised a number of different principles of artificial pancreas in an attempt to solve this problem. Renard, Eric et al (Renard E, Costalat G, Chevasssus H, Bringer J. closed loop insulin delivery expressed insulin pumps and sensors in Type 1 diabetes drugs. diabetes Res. clin. Practer. 74, S173-S177 (2006)) use an implanted insulin pump based on a coupling model to control insulin infusion via the peritoneal pathway as well as the glucose sensor in the vein. Due to insufficient sensor performance, the sensor stops working 24 hours after being implanted, and effectively keeps the glucose within 80-240mg/dl for 84.1 percent of the time during the working period. Ikeda H et al (Ikeda H, Kobayashi N, Tanaka Y, et al, Anewly depleted biological tissue culture sugar in total culture tissue culture. tissue Eng.2006 Jul; 12(7):1799 and 809) developed a novel bone-specific alkaline phosphatase (BAP) of glucose and insulin consisting of polyethylene-vinyl alcohol (EVAL) hollow fibers and a polyurethane-coated non-woven Polytetrafluoroethylene (PTFE) cell adhesion. The porcine islets adhere to the surface of the PTFE fabric, rather than to the surface of the EVAL hollow fibers, allowing nutrient and oxygen exchange between the fibers and the blood flowing outside the cells. It inoculates this BAP with porcine islets and links it into the circulation of fully pancreas-excised diabetic pigs. The blood sugar level is reduced to a normal range, the health condition is greatly improved, and the survival time is prolonged.
The patent CN104958077A intelligent control closed-loop artificial pancreas system calculates the dosage of the real-time insulin infusion device by receiving the human blood sugar monitoring data of the continuous blood sugar monitoring device in real time and utilizing a built-in fuzzy self-adaptive proportional-calculus control algorithm according to the real-time human blood sugar monitoring data, but needs portable external equipment and regularly replaces catheters, brings great inconvenience to the daily life of a patient, and charges a battery of an implanted device, and the problems of long-term insulin stability and the like in the implanted reservoir are difficult to solve. The bioartificial pancreas including sugar-sensitive microcapsules has the disadvantages of immune rejection, difficult shape and size matching with the defective pancreas when implanted, and the like.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for constructing an artificial intelligence pancreas for reducing blood sugar based on 3D printing.
The invention aims to construct an artificial intelligent pancreas for reducing blood sugar by utilizing 3D printing, and overcomes the defects that the conventional blood sugar reducing means needs to detect blood sugar for multiple times for subcutaneous injection, and the existing human pancreas needs to carry external equipment and replace accessories.
The invention constructs 'modified gelatin-sodium alginate-acetalized dextran microspheres' three-in-one hydrogel, disperses glucose oxidase into the hydrogel, and entraps insulin into the acetalized dextran microspheres as an intelligent response type release system. When the blood sugar concentration is increased, the glucose oxidase oxidizes the glucose into gluconic acid, the pH value of the place is reduced, the acid-sensitive acetalized dextran microspheres are rapidly degraded to release insulin, the blood sugar is further reduced, and multiple blood sugar detection and subcutaneous injection are avoided.
Through biological 3D printing advantage lies in can be according to the actual conditions that the pancreas is defected or amputated, print out the artificial pancreas that has fine structure and corresponding shape size to agree with impaired pancreas better. The used composite hydrogel has enough strength, the materials such as sodium alginate and gelatin have good biocompatibility and biodegradability, and the degradation speed can be controlled by adjusting the proportion according to needs, so that the defects that the battery, the catheter and the like need to be replaced in the conventional artificial pancreas are overcome. The corresponding relation between the glucose concentration and the insulin release can be adjusted by changing the amount of the entrapped glucose oxidase and controlling the conversion rate of hydroxyl in the acetalization reaction of the glucan, so that the aim of controlling the blood glucose concentration in a normal range for a long time is fulfilled.
The purpose of the invention is realized by the following technical scheme:
a method for constructing an artificial intelligence pancreas for reducing blood glucose based on 3D printing, comprising the steps of:
(1) preparation of acetalized dextran loaded insulin nano particle
Firstly, dissolving insulin in dilute hydrochloric acid, stirring for a certain time, adjusting the pH value to be about 3.0, and dissolving to obtain an insulin solution; dissolving acetalized dextran into dichloromethane, adding into insulin solution, and performing ultrasonic treatment to obtain emulsion; adding the emulsion into 3% w/w PVA solution (in PBS, MW 13000-; then immediately pouring the obtained double emulsion into a second PVA solution (10mL, 0.3% w/w PBS), stirring, evaporating the organic solvent, centrifuging, washing with PBS (once) and double distilled water (twice), centrifuging after vortex ultrasonic treatment to remove supernatant, finally resuspending with double distilled water, and freeze-drying to obtain acetalized dextran-loaded insulin nanoparticles;
(2) preparation of hydrogel for printing
Preparing 2-6 percent (preferably 4.5 percent) of light-cured gelatin and 1-5 percent (preferably 3 percent) of sodium alginate, magnetically stirring the mixture evenly, performing ultraviolet sterilization treatment, adding 0.1 percent of Irgacure 2959 photoinitiator, and fully mixing the mixture to prepare gelatin and sodium alginate mixture solution; dissolving glucose oxidase into physiological saline before printing, adding the glucose oxidase and acetalized dextran loaded insulin nanoparticles into a gelatin-sodium alginate mixture solution, and fully and uniformly stirring to prepare hydrogel for printing;
(3) preparation of hydrogel support baths
The hydrogel supporting bath is made of temperature-sensitive gelatin, and the preparation method comprises the following steps: firstly, using CaCl2Preparing 4.5% (w/v) gelatin solution, placing in a jar at 4-8 deg.C for 12-24 hr (preferably 4 deg.C for 12 hr), and adding CaCl at 4 deg.C2Dissolving, fully crushing gelatin granules; centrifuging to remove supernatant, and adding CaCl at 4 deg.C2The solution resuspension was continued until no air bubbles were observed at the top of the supernatant, indicating that most of the soluble gelatin was removed, making a hydrogel support bath, stored at 4 ℃; when in use, the liquid is introduced into a container, and the wiping paper is used for absorbing the redundant liquid;
(4)3D printing preparation
Extracting a pancreas 3D model from the MRI image, importing the pancreas 3D model into 3D printing software, and slicing; loading the supporting bath gelatin into the transparent container used, and aspirating off excess liquid; centrifuging hydrogel for printing to remove bubbles, loading the hydrogel into a printing head, and printing pancreas; because the supporting bath gelatin contains calcium ions, the sodium alginate is solidified in the printing process, and after the printing is finished, the printing pancreas is subjected to double cross-linking molding by using 365nm ultraviolet lamp with power of 800mW at a position of 5-10 cm (preferably 7cm) for 30-60 seconds (preferably 30 seconds); heating in a 37 +/-1 ℃ water bath to melt the gelatin of the support bath and obtain the printed artificial intelligent pancreas. The printer adopts an extrusion type biological 3D printer, and the printing temperature is set to be 37 +/-1 ℃; the printing speed is 15 mm/s-25 mm/s; the inner diameter of the printing needle head is 0.1-0.25 mm; the internal printing pressure is 0.2-0.5 KPa.
In the step (1), the step (c),
preferably, the dilute hydrochloric acid is 1mmol/L HCl (pH 3.0).
Preferably, the pH is adjusted to about 3.0 by 10mmol/L HCl (pH 2.0).
Preferably, the concentration of insulin in the insulin solution is 200-400 mg/mL; more preferably 200 mg/mL.
Preferably, the mass ratio of the acetalized glucan to the insulin is 10: 1-20: 1; more preferably 20: 1.
Preferably, the ultrasonic condition is 30W ultrasonic for 5-10 s; more preferably 30W ultrasound for 5 s.
Preferably, the ice bath ultrasound condition is 30W ice bath ultrasound for 30-60 s; more preferably 30W ice bath ultrasound for 30 s.
Preferably, the stirring time is 3-4 h; more preferably 3 h.
Preferably, the centrifugation condition is 14800g, and the centrifugation is carried out for 15-20 min at 4 ℃; more preferably 14800g, centrifuged at 4 ℃ for 15 min.
Preferably, the second PVA solution is a 0.3% w/w PVA solution.
In the step (2),
preferably, the light-curable gelatin is GelMA (methacrylate gelatin).
Preferably, the concentration of the glucose oxidase in physiological saline is 100 mg/mL.
Preferably, the mass ratio of the glucose oxidase to the acetalized dextran-loaded insulin nanoparticles is 4: 1-1: 1; more preferably 1: 1.
In the step (3), the step (c),
preferably, said CaCl2The solution was 11mM CaCl2And (3) solution.
Preferably, the stirring time is 120s to 240 s; more preferably 165 s.
Preferably, the centrifugation condition is 4200rpm centrifugation for 2-4 min; more preferably, centrifugation is carried out at 4200rpm for 2 min.
In the step (4), the step (c),
preferably, the centrifugation condition is 800-1000 rpm centrifugation for 1 min; more preferably at 800rpm for 1 min.
Preferably, the printing speed is 15 mm/s; the inner diameter of the printing needle is 0.25 mm; the internal printing pressure was 0.5 KPa.
The mechanism of the invention is as follows:
the artificial intelligent pancreas for reducing the blood sugar is constructed based on 3D printing, and the release of insulin can be controlled according to the real-time blood sugar concentration, so that the aim of intelligently reducing the blood sugar is fulfilled. The 3D printing technology can print out the artificial pancreas with fine structure and corresponding shape and size according to the actual condition of pancreas defect or excision to better agree with impaired pancreas. Compared with the existing artificial pancreas, the invention does not need to carry portable equipment, does not need to replace batteries, catheters and the like, can greatly reduce the burden of patients, and has outstanding value of application basic research and market development prospect with great potential.
Compared with the prior art, the invention has the following advantages and effects:
the artificial intelligent pancreas prepared by 3D printing has the advantages of good biocompatibility and biodegradability, and does not need to carry external equipment or replace a battery catheter. The commonly used blood sugar concentration measurement and subcutaneous insulin injection for many times bring great pain and inconvenience to patients, in addition, the injected insulin has the defects of unstable absorption, peak concentration, incapability of well simulating insulin secretion, difficulty in accurately calculating the injection amount according to the absorption level of the patients, possibility of causing hypoglycemia due to calculation errors and the like. The modified gelatin-sodium alginate-acetalized dextran microspheres' three-in-one hydrogel-glucose oxidase intelligent blood glucose concentration response type release system can well avoid the problems. The corresponding relation between the glucose concentration and the insulin release can be adjusted by changing the amount of the entrapped glucose oxidase and controlling the conversion rate of hydroxyl in the acetalization reaction of the glucan, so that the aim of controlling the blood glucose concentration in a normal range for a long time is fulfilled.
Patent 201610830529.5 carries out three-dimensional printing and in-vitro culture of brain tumor in-vitro models by directly mixing hyaluronic acid, sodium alginate, gelatin, glutamine transaminase and brain tumor cells, but with the improvement of the number of printing layers, the supporting strength is insufficient, the tumor tissue structure cannot be well reduced, and the common 3D biological printing method is not ideal in the treatment of space configuration and internal structure. Compared with the hydrogel direct printing, the suspension printing technology adopted by the invention can avoid the collapse problem caused by insufficient mechanical strength of the hydrogel when the number of printing layers is higher, and can print a more complex suspension structure. Compared with a pancreas model manufactured by a traditional method, the biological 3D suspension printing method has the advantages that the artificial pancreas with a fine structure and a corresponding shape and size can be printed according to the actual condition of pancreas defect or excision, so that the artificial pancreas can be better fitted with the damaged pancreas. The used composite hydrogel has enough strength, and the materials such as sodium alginate, gelatin and the like have good biocompatibility and biodegradability, and the degradation speed can be controlled by adjusting the proportion of the materials according to the requirement.
Drawings
FIG. 1 is a schematic flow chart of the present invention for constructing an artificial intelligence pancreas for reducing blood glucose based on 3D printing.
FIG. 2 is a schematic diagram of the present invention building an artificial intelligence pancreas for hypoglycemic purposes based on 3D printing; wherein, A: a biological 3D printer; b: a 3D model of the pancreas; c, D: gel 3D printing pancreas.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The invention discloses a flow chart of constructing an artificial intelligence pancreas for reducing blood sugar based on 3D printing, which is shown in figure 1.
Example 1 preparation of an Intelligent hypoglycemic Artificial pancreas
(1) Preparation of acetalized dextran loaded insulin nano particle
First, 10mg of insulin was dissolved in 1mmol/L HCl (pH 3.050 μ L), stirred for a certain period of time, and then adjusted to pH 3.0 with 10mmol/L HCl (pH 2.0) to be dissolved, thereby obtaining an insulin solution. 200mg of the acetalized dextran was dissolved in 1mL of dichloromethane, added to the insulin solution, and subjected to ultrasonic treatment with an ultrasonic probe at 30W for 5 seconds to prepare an emulsion. This emulsion was added to a 3% W/W PVA solution (in PBS, MW 13,000-23,000g/mol, 87-89% hydroszed) and sonicated in a 30W ice bath for 30s to produce a double emulsion. The resulting double emulsion was then immediately poured into a second PVA solution (10mL, 0.3% w/w PBS), stirred for 3 hours to evaporate the organic solvent, centrifuged at 14800g at 4 ℃ for 15min, washed with 50mL PBS (once) and double distilled water (twice), centrifuged after vortexing to remove the supernatant, finally resuspended in double distilled water (2mL, pH 8.0), lyophilized to produce acetalized dextran loaded insulin nanoparticles.
(2) Preparation of hydrogel for printing
Preparing GelMA (light-cured gelatin) 4.5 percent and sodium alginate 3 percent, magnetically stirring uniformly, performing ultraviolet sterilization treatment, adding 0.1 percent Irgacure 2959 photoinitiator, and fully mixing to prepare a gelatin sodium alginate mixture solution; before printing, 10mg of glucose oxidase is dissolved into 100 mu L of physiological saline, then the glucose oxidase and 10mg of acetalized dextran loaded insulin nanoparticles are added into the gelatin-sodium alginate mixture solution together, and the mixture is fully and uniformly stirred to prepare the hydrogel for printing.
(3) Preparation of a support bath for hydrogels
The hydrogel supporting bath is made of temperature-sensitive gelatin, and the preparation method comprises the following steps: with 11mM CaCl2The solution was prepared as 150mL of 4.5% (w/v) gelatin solution, placed in a 500mL jar at 4 ℃ for 12h, followed by the addition of 350 mL of 11mM CaCl at 4 DEG2The solution was stirred with a home grade juicer for 165s to break up the gelatin particles thoroughly. Then, the mixture was put into a 50mL centrifuge tube and centrifuged at 4200rpm for 2 min. The supernatant was removed and washed with 11mM CaCl at 4 deg.C2Solution replacement, resuspension continued until no air bubbles were observed at the top of the supernatant, indicating that most of the soluble gelatin was removed. Storing at 4 ℃. In use, the liquid is introduced into a container, and the wiping paper is used for absorbing excessive liquid.
(4)3D printing preparation
A3D pancreas model is extracted from the MRI image, introduced into 3D printing software, and sliced. The supporting bath gelatin was loaded into the transparent container used and the excess liquid was aspirated. The printing hydrogel was centrifuged at 800rpm for 1min to remove air bubbles, and then loaded into a print head to perform printing of the pancreas. Because the gelatin of the supporting bath contains calcium ions, the sodium alginate is solidified in the printing process, and after the printing is finished, the double-crosslinking formation of the printed pancreas is finished by crosslinking 30s at a position of 7cm with the power of a 365nm ultraviolet lamp of 800 mW. Heating in a 37 ℃ water bath to melt the gelatin of the support bath and obtain the printed artificial intelligent pancreas. The printer adopts an extrusion type biological 3D printer, and the printing temperature is set to be 37 ℃; the printing speed is 15 mm/s; the inner diameter of the printing needle is 0.25 mm; the internal printing pressure was 0.5 KPa.
The embodiment is a schematic diagram of constructing an artificial intelligence pancreas for reducing blood sugar based on 3D printing, as shown in fig. 2, wherein a: a biological 3D printer; b: a 3D model of the pancreas; c and D: gel 3D printing pancreas. As can be seen from fig. 2, the artificial intelligence pancreas with actual pancreas morphology can be printed out from the 3D image by using the method of the present invention, and can be well matched with the defective pancreas.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (10)

1. A method for constructing an artificial intelligence pancreas for reducing blood glucose based on 3D printing is characterized by comprising the following steps:
(1) preparation of acetalized dextran loaded insulin nano particle
Firstly, dissolving insulin in dilute hydrochloric acid, stirring for a certain time, adjusting the pH value to 3.0, and dissolving to obtain an insulin solution; dissolving acetalized dextran into dichloromethane, adding into insulin solution, and performing ultrasonic treatment to obtain emulsion; adding the emulsion into a 3% w/w PVA solution, and carrying out ice bath ultrasonic treatment to obtain double emulsion; then immediately pouring the obtained double emulsion into a second PVA solution, stirring to evaporate an organic solvent, centrifuging, washing once with PBS and twice with double distilled water, centrifuging after vortex ultrasonic treatment to remove a supernatant, finally resuspending with double distilled water, and freeze-drying to prepare acetalized dextran loaded insulin nanoparticles;
(2) preparation of hydrogel for printing
Preparing 2-6% of light-cured gelatin and 1-5% of sodium alginate, magnetically stirring uniformly, performing ultraviolet sterilization treatment, adding 0.1% of Irgacure 2959 photoinitiator, and fully mixing to prepare a gelatin and sodium alginate mixture solution; dissolving glucose oxidase into physiological saline before printing, adding the glucose oxidase and acetalized dextran loaded insulin nanoparticles into a gelatin-sodium alginate mixture solution, and fully and uniformly stirring to prepare hydrogel for printing;
(3) preparation of hydrogel support baths
The hydrogel supporting bath is made of temperature-sensitive gelatin, and the preparation method comprises the following steps: firstly, using CaCl2Preparing 4.5% gelatin solution, placing in a jar at 4-8 deg.C for 12-24 hr, and adding CaCl at 4 deg.C2Dissolving, fully crushing gelatin granules; centrifuging to remove supernatant, and adding CaCl at 4 deg.C2The solution resuspension was continued until no air bubbles were observed at the top of the supernatant, indicating that most of the soluble gelatin was removed, making a hydrogel support bath, stored at 4 ℃; when in use, the liquid is introduced into a container, and the wiping paper is used for absorbing the redundant liquid;
(4)3D printing preparation
Extracting a pancreas 3D model from the MRI image, importing the pancreas 3D model into 3D printing software, and slicing; loading the supporting bath gelatin into the transparent container used, and aspirating off excess liquid; centrifuging hydrogel for printing to remove bubbles, loading the hydrogel into a printing head, and printing pancreas; because the supporting bath gelatin contains calcium ions, the sodium alginate is solidified in the printing process, and after printing is finished, double cross-linking forming of the printed pancreas is finished by cross-linking at a position of 5-10 cm for 30-60 seconds by using 365nm ultraviolet lamp power of 800 mW; heating in a water bath at 37 +/-1 ℃ to melt gelatin in the supporting bath to obtain the printed artificial intelligent pancreas; the printer adopts an extrusion type biological 3D printer, and the printing temperature is set to be 37 +/-1 ℃; the printing speed is 15 mm/s-25 mm/s; the inner diameter of the printing needle head is 0.1-0.25 mm; the internal printing pressure is 0.2-0.5 KPa.
2. The method for constructing an artificial intelligence pancreas for lowering blood glucose based on 3D printing according to claim 1, wherein:
in the step (1), the step (c),
the concentration of insulin in the insulin solution is 200-400 mg/mL;
the mass ratio of the acetalized glucan to the insulin is 10: 1-20: 1.
3. The method for constructing an artificial intelligence pancreas for lowering blood glucose based on 3D printing according to claim 1, wherein:
in the step (1), the step (c),
the ultrasonic condition is 30W ultrasonic for 5-10 s;
the ice bath ultrasound condition is 30W ice bath ultrasound for 30-60 s.
4. The method for constructing an artificial intelligence pancreas for lowering blood glucose based on 3D printing according to claim 1, wherein:
in the step (1), the step (c),
the stirring time is 3-4 h;
the centrifugation condition is 14800g, and the centrifugation is carried out for 15-20 min at 4 ℃.
5. The method for constructing an artificial intelligence pancreas for lowering blood glucose based on 3D printing according to claim 1, wherein:
the second PVA solution was a 0.3% w/w PVA solution.
6. The method for constructing an artificial intelligence pancreas for lowering blood glucose based on 3D printing according to claim 1, wherein:
in the step (2),
the light-cured gelatin is GelMA;
the concentration of the glucose oxidase in physiological saline is 100 mg/mL.
7. The method for constructing an artificial intelligence pancreas for lowering blood glucose based on 3D printing according to claim 1, wherein:
the mass ratio of the glucose oxidase to the acetalized dextran-loaded insulin nanoparticles is 4: 1-1: 1.
8. The method for constructing an artificial intelligence pancreas for lowering blood glucose based on 3D printing according to claim 1, wherein:
in the step (3), the step (c),
the stirring time is 120-240 s;
the centrifugation condition is centrifugation for 2-4 min at the rotating speed of 4200 rpm.
9. The method for constructing an artificial intelligence pancreas for lowering blood glucose based on 3D printing according to claim 1, wherein:
in the step (4), the step (c),
the centrifugation condition is centrifugation for 1min at the rotating speed of 800-1000 rpm.
10. The method for constructing an artificial intelligence pancreas for lowering blood glucose based on 3D printing according to claim 1, wherein:
the printing speed is 15 mm/s; the inner diameter of the printing needle is 0.25 mm; the internal printing pressure was 0.5 KPa.
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