CN115558127A - Preparation and application of 4D printing chitosan-based cell carrier - Google Patents
Preparation and application of 4D printing chitosan-based cell carrier Download PDFInfo
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- CN115558127A CN115558127A CN202211011551.9A CN202211011551A CN115558127A CN 115558127 A CN115558127 A CN 115558127A CN 202211011551 A CN202211011551 A CN 202211011551A CN 115558127 A CN115558127 A CN 115558127A
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- sodium alginate
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- 229920001661 Chitosan Polymers 0.000 title claims abstract description 82
- 238000002360 preparation method Methods 0.000 title claims abstract description 27
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 claims description 44
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- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 39
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- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
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Abstract
The invention belongs to the technical field of production and preparation of medical materials, and relates to preparation and application of a 4D printing chitosan-based cell carrier.
Description
The technical field is as follows:
the invention belongs to the technical field of production and preparation of medical materials, and relates to preparation and application of a 4D printing chitosan-based cell carrier, in particular to hydroxyethyl chitosan temperature-sensitive gel for repairing DFU prepared on the basis of a 4D printing technology.
The background art comprises the following steps:
diabetes Mellitus (DM) is a common chronic metabolic disease caused by hyposecretion or hypofunction of insulin, and is clinically characterized by hyperglycemia, which can cause metabolic disorders of proteins, fats and carbohydrates. Studies report that the prevalence of diabetes is expected to rise from 2.85 billion in 2010 to more than 4.39 billion in 2030.
Long-term hyperglycemia stimulation causes elevation of Glycated Hemoglobin (GHb), advanced Glycation End Products (AGEs), and the like, causes severe macrovascular and microvascular lesions, can also cause Diabetic neuropathy, and the condition is irreversible, and finally induces Diabetic skin ulcers, such as Diabetic leg ulcers, foot ulcers (DFU), and the like. Studies have shown that approximately 25% of diabetic patients suffer from DFU, require repeated surgical intervention, have become a leading cause of non-traumatic amputation, and have a 5-year mortality rate of > 50%, placing a tremendous economic and psychological burden on the patient.
The DFU can be used for comprehensive treatment of surgery, medicines, nursing, feet and the like, so that the amputation rate of diabetic feet is reduced, and meanwhile, health education related to diseases needs to be given to patients, wherein the health education related to the diseases comprises foot decompression modes, foot treatment, surgical debridement, anti-infection treatment, blood flow reconstruction and the like. Furthermore, the patient needs to be controlled for glycated hemoglobin values <7% to promote wound healing. If the patient has difficult-to-heal injury and extensive tissue defect, amputation below the knee of the patient is needed besides blood flow reconstruction, so that the life quality of the patient is deteriorated, and the life of the patient can be threatened in severe cases.
The prior art mainly reduces the risk of cardiovascular diseases by a blood sugar reducing mode, but the risk of the cardiovascular diseases developing into microangiopathy still exists. Finding efficient processing and intervention methods is a preoccupation to solve DFUs.
The process of the preparation method of the 4D-chitosan temperature-sensitive gel disclosed by the Chinese patent 201910871003.5 comprises the following steps:
(1) Weighing chitosan, dissolving the chitosan in an acetic acid aqueous solution at room temperature, stirring until the chitosan is completely dissolved to obtain a chitosan solution, printing the chitosan solution into chitosan temperature-sensitive gel with the pore diameter of 50-100 mu m by adopting a 4D bio-printer according to set printing parameters, enabling the chitosan temperature-sensitive gel to form a set shape structure, and performing freeze drying after solvent is removed to obtain freeze-dried chitosan;
(2) Adding ultrapure water and beta-sodium glycerophosphate into a water bath kettle at the temperature of 60-70 ℃, and naturally cooling to room temperature after the beta-sodium glycerophosphate is dissolved to obtain a beta-sodium glycerophosphate water solution;
(3) Preparing a carboxymethyl chitosan aqueous solution by using ultrapure water at room temperature, dropwise adding the beta-sodium glycerophosphate aqueous solution obtained in the step (2) into the carboxymethyl chitosan aqueous solution, and uniformly mixing to obtain a mixed solution;
(4) And (2) crosslinking the freeze-dried chitosan obtained in the step (1) with the mixed solution obtained in the step (3) for 1-2 minutes to obtain the 4D-chitosan temperature-sensitive gel with uniform pore size. The prepared 4D-chitosan temperature-sensitive gel is coated with limbal stem cells and then is applied to the surface of an alkali-burned cornea, and the limbal stem cells are released to repair the wound surface.
The hydroxyethyl chitosan temperature-sensitive gel for repairing DFU is researched and designed based on the 4D printing technology, and has economic and social benefits.
The invention content is as follows:
the invention aims to overcome the defects in the prior art, and researches and designs the preparation and application of a 4D printing chitosan-based cell carrier, so as to prepare the 4D printing chitosan-based cell carrier which can effectively improve the entrapment efficiency of adipose-derived mesenchymal stem cells and promote skin reconstruction.
In order to achieve the purpose, the preparation method of the 4D printing chitosan-based cell carrier comprises four steps of preparing sodium periodate aqueous solution, preparing sodium alginate oxide, preparing hydroxyethyl chitosan in-situ hydrogel and preparing the 4D printing chitosan-based cell carrier:
(1) Preparation of aqueous sodium periodate solution
Dissolving sodium periodate powder in deionized water at normal temperature to prepare a sodium periodate aqueous solution with the mass percentage concentration of 2%;
(2) Preparation of oxidized sodium alginate
Under the condition of normal temperature and stirring, adding a sodium periodate aqueous solution into a sodium alginate aqueous solution with the mass percentage concentration of 2% to form a mixed solution, carrying out oxidation reaction, after 24 hours, adding 100 mu L of ethylene glycol into the mixed solution to stop the oxidation reaction, after 0.5 hour, carrying out suction filtration on the mixed solution, dehydrating and precipitating with absolute ethyl alcohol to obtain a white solid, drying the white solid in vacuum at low temperature to obtain an oxidized sodium alginate crude product, dissolving the oxidized sodium alginate crude product into an oxidized sodium alginate crude aqueous solution with the mass percentage concentration of 2%, placing the oxidized sodium alginate crude aqueous solution into a dialysis bag for dialysis at the temperature of 4 ℃, changing 1 time of distilled water every 6 hours, after complete dialysis, centrifuging, taking supernatant, and freeze-drying to obtain the oxidized sodium alginate with the oxidation degree of 10%;
the molar ratio of sodium alginate to sodium periodate is 10;
(3) Preparation of hydroxyethyl chitosan in situ hydrogel
Mixing hydroxyethyl chitosan with the mass percentage concentration of 2% and oxidized sodium alginate according to the mass ratio of 8;
(4) Preparation of 4D-printed Chitosan-based cell Carrier
And (3) printing the hydroxyethyl chitosan in-situ hydrogel into hydroxyethyl chitosan temperature-sensitive gel with the aperture of 50-100 mu m and a set shape structure by adopting a 4D bioprinter according to set printing parameters, removing the solvent, and freeze-drying to obtain the 4D printing chitosan-based cell carrier with uniform aperture size and capable of being stored for 6-12 months in a sterile environment at the temperature of 4-15 ℃.
The 4D printed chitosan-based cell carrier prepared by the method is pasted on the surface of the diabetic ulcer skin after being coated with the adipose-derived mesenchymal stem cells, and the adipose-derived mesenchymal stem cells are released to repair the wound surface; can also be used for repairing affected parts such as skin burn, severe injury of cornea and cervix, etc.
Compared with the prior art, firstly, dissolving sodium periodate powder in deionized water to prepare sodium periodate aqueous solution, then stirring the sodium alginate aqueous solution and the sodium periodate aqueous solution to form mixed solution, carrying out oxidation reaction, adding ethylene glycol to stop the oxidation reaction, filtering the mixed solution, dehydrating and precipitating with absolute ethyl alcohol, carrying out low-temperature vacuum drying to prepare crude sodium alginate oxide, dissolving the crude sodium alginate oxide aqueous solution into distilled water, carrying out dialysis, centrifuging to obtain supernatant, carrying out freeze drying to prepare sodium alginate oxide, finally mixing hydroxyethyl chitosan and sodium alginate oxide, carrying out cross-linking reaction, extruding and pushing into a test tube to obtain hydroxyethyl chitosan in-situ hydrogel, printing the hydroxyethyl chitosan in-situ hydrogel by using a 4D biological printer, removing a solvent, carrying out freeze drying to obtain a 4D printed chitosan-based cell carrier, being capable of being used as a transplantation carrier of adipose-derived mesenchymal stem cells, effectively improving the entrapment efficiency of the adipose-derived stem cells, and promoting the reconstruction of diabetic ulcer skin; the principle is scientific and reliable, the 4D printing chitosan-based cell carrier which is prepared by the 4D bioprinting technology, has consistent pore size and a set shape structure improves the entrapment efficiency and capacity of the adipose-derived mesenchymal stem cells, has important theoretical significance and wide application prospect in the field of DFU repair, provides help for DFU patients in clinical medicine, and improves the life quality of the DFU patients.
Description of the drawings:
fig. 1 is a schematic structural diagram of a 4D printed chitosan-based cell carrier prepared in example 1 of the present invention.
Fig. 2 is a schematic diagram of a state in which adipose-derived mesenchymal stem cells are encapsulated in a 10-fold under-the-scope 4D printed chitosan-based cell carrier according to embodiment 2 of the present invention.
Fig. 3 is a schematic comparison view of effects of a traditional temperature-sensitive gel-encapsulated adipose-derived mesenchymal stem cell and a 4D-printed chitosan-based cell carrier-encapsulated adipose-derived mesenchymal stem cell on repairing a wound surface of a diabetic mouse according to embodiment 3 of the present invention.
The specific implementation mode is as follows:
the invention is further illustrated by the following examples in connection with the accompanying drawings.
Example 1:
the preparation method of the 4D printing chitosan-based cell carrier comprises the following four steps of preparing a sodium periodate aqueous solution, preparing sodium alginate oxide, preparing hydroxyethyl chitosan glycogen into hydrogel and preparing hydroxyethyl chitosan in-situ hydrogel:
(1) Preparation of aqueous sodium periodate solution
Dissolving 1g of sodium periodate powder in 50ml of deionized water at normal temperature to obtain a sodium periodate aqueous solution with the mass percentage concentration of 2%;
(2) Preparation of oxidized sodium alginate
Adding a sodium periodate aqueous solution into a sodium alginate aqueous solution with the mass percentage concentration of 2% under the conditions of normal temperature and stirring to form a mixed solution, carrying out oxidation reaction, after 24 hours, adding 100 mu L of ethylene glycol into the mixed solution to stop the oxidation reaction, after 0.5 hour, carrying out suction filtration on the mixed solution, dehydrating and precipitating with absolute ethyl alcohol to obtain a white solid matter, drying the white solid matter in vacuum at low temperature to obtain an oxidized sodium alginate crude product, dissolving the oxidized sodium alginate crude product into an oxidized sodium alginate crude product aqueous solution with the mass percentage concentration of 2%, placing the oxidized sodium alginate crude product aqueous solution into a dialysis bag for dialysis at the temperature of 4 ℃, changing distilled water for 1 time every 6 hours, determining that the oxidized sodium alginate crude product aqueous solution is completely dialyzed by using a conductivity meter, centrifuging, taking a supernatant, and freeze-drying to obtain oxidized sodium alginate with the oxidation degree of 10%;
the molar ratio of sodium alginate to sodium periodate is 10;
(3) Preparation of hydroxyethyl Chitosan in-situ hydrogel
Mixing hydroxyethyl chitosan with the mass percentage concentration of 2% and oxidized sodium alginate in an equal volume manner according to the mass ratio of 8;
(4) Preparation of 4D-printed Chitosan-based cell Carrier
Adopting a UN-4DBI-C01 type 4D bioprinter to print the following components according to set printing parameters: the pore spacing is 600 μm, the layer thickness is 100 μm, the platform temperature is-25 ℃, the printing speed is 15mm/s, the nozzle diameter is 0.3mm, the discharge amount is 0.1g/min, the hydroxyethyl chitosan in-situ hydrogel is printed into a round hydroxyethyl chitosan temperature-sensitive gel with the pore diameter of 50-100 μm, and the 4D printing chitosan-based cell carrier shown in figure 1 is obtained by removing the solvent and freeze drying.
Example 2:
the process of the 4D printed chitosan-based cell carrier encapsulated adipose-derived mesenchymal stem cells prepared in the embodiment 1 comprises the following steps:
firstly, placing a 4D printing chitosan-based cell carrier in a cell culture dish for full infiltration to obtain the 4D printing chitosan-based cell carrier to be coated;
then, separating and culturing adipose-derived mesenchymal stem cells and adding the adipose-derived mesenchymal stem cells into a 4D printing chitosan-based cell carrier to be encapsulated;
finally, after 24h of culture, the state of the adipose-derived mesenchymal stem cells encapsulated by the 4D printed chitosan-based cell carrier was observed under a microscope with a 10-fold microscope, and the results are shown in fig. 2.
Example 3:
the embodiment relates to a contrast test of the effect of repairing the wound surface of a diabetic mouse by a 4D printing chitosan-based cell carrier and a traditional temperature-sensitive gel-encapsulated adipose-derived mesenchymal stem cell:
(1) Establishing a diabetes mouse model, and modeling the skin of a mouse after 8 weeks;
(2) Anesthetizing a mouse by using chloral hydrate with the mass percentage concentration of 3%;
(3) Shaving the mice, and carrying out deep depilation on the mice through the depilatory cream;
(4) Disinfecting the skin of the mouse by using iodophor;
(5) In a biological safety cabinet, a 5mm skin sampler is used for forming a full-layer skin wound surface on the back skin of a mouse to obtain a skin wound surface model of the diabetic mouse, and iodophors are used for disinfecting the wound surface skin;
(6) Treating the skin wound model of the diabetic mouse obtained in the step (5) by using PBS to obtain a PBS treatment model;
(7) Repairing the skin wound model of the diabetic mouse obtained in the step (5) by using the traditional temperature-sensitive gel-loaded adipose-derived mesenchymal stem cells to obtain a traditional temperature-sensitive gel repair model;
(8) Repairing the diabetic mouse skin wound model obtained in the step (5) by using the 4D printing chitosan-based cell carrier prepared in the embodiment 1 to load the angular adipose mesenchymal stem cells to obtain a 4D printing chitosan-based cell carrier repairing model;
(9) Respectively adding 1% double antibody by mass percentage into drinking water of the mouse repaired in the steps (6), (7) and (8) to prevent infection, wrapping the wound surface of the mouse with gauze, keeping warm, measuring the wound surface area in 0, 4, 7, 10 and 14 days, and taking a picture for recording; the result is shown in fig. 3, after the skin wound of the diabetic mouse is repaired, the area of the skin wound after the repair in steps (6), (7) and (8) is gradually reduced along with the passage of time, wherein the repair effect of the 4D printing chitosan-based cell carrier is obviously superior to that of the traditional temperature sensitive gel and PBS treatment group.
Claims (9)
1. A preparation method of a 4D printing chitosan-based cell carrier comprises four steps of preparing sodium periodate aqueous solution, preparing oxidized sodium alginate, preparing hydroxyethyl chitosan in-situ hydrogel and preparing the 4D printing chitosan-based cell carrier, and is characterized in that:
(1) Preparation of aqueous sodium periodate solution
Dissolving sodium periodate powder in deionized water at normal temperature to obtain sodium periodate aqueous solution;
(2) Preparation of oxidized sodium alginate
Adding a sodium periodate aqueous solution into a sodium alginate aqueous solution under the conditions of normal temperature and stirring to form a mixed solution, adding ethylene glycol into the mixed solution to terminate oxidation reaction after the mixed solution is subjected to oxidation reaction, then carrying out suction filtration on the mixed solution and carrying out dehydration and precipitation by absolute ethyl alcohol to obtain a white solid matter, drying the white solid matter in vacuum at low temperature to obtain an oxidized sodium alginate crude product, dissolving the oxidized sodium alginate crude product into an oxidized sodium alginate crude product aqueous solution by distilled water and dialyzing, centrifuging after dialysis is completed, taking supernatant and freeze-drying to obtain oxidized sodium alginate;
(3) Preparation of hydroxyethyl chitosan in situ hydrogel
Mixing hydroxyethyl chitosan and oxidized sodium alginate, and performing crosslinking reaction to obtain hydroxyethyl chitosan in-situ hydrogel;
(4) Preparation of 4D-printed Chitosan-based cell Carrier
And (3) printing the hydroxyethyl chitosan in-situ hydrogel into hydroxyethyl chitosan temperature-sensitive gel with the aperture of 50-100 microns and a set shape structure by adopting a 4D bioprinter according to set printing parameters, removing the solvent, and freeze-drying to obtain the 4D printed chitosan-based cell carrier with uniform aperture size and capable of being stored for 6-12 months in a sterile environment at the temperature of 4-15 ℃.
2. The method for preparing a 4D-printed chitosan-based cell carrier according to claim 1, wherein the concentration of the sodium periodate aqueous solution obtained in step (1) is 2% by mass.
3. The method for preparing a 4D-printed chitosan-based cell carrier according to claim 1, wherein the mass percentage concentration of the sodium alginate aqueous solution involved in the step (2) is 2%.
4. The method for preparing a 4D printed chitosan-based cell carrier according to claim 3, wherein the molar ratio of sodium alginate to sodium periodate is 10.
5. A4D printed chitosan-based cell carrier preparation method according to claim 4, wherein the time of the oxidation reaction is 24h, the volume of the ethylene glycol is 100 μ L, and the mixed solution is filtered with suction after 0.5h of the oxidation reaction; the mass percentage concentration of the oxidized sodium alginate crude product water solution is 2 percent; the dialysis was carried out at a temperature of 4 ℃ with 1 change of distilled water every 6 hours.
6. A4D-printed chitosan-based cell carrier preparation method according to claim 5, wherein the oxidation degree of the oxidized sodium alginate obtained in step (2) is 10%.
7. The preparation method of the 4D printing chitosan-based cell carrier according to claim 1, wherein the mass percentage concentration of the hydroxyethyl chitosan involved in the step (3) is 2%, and the hydroxyethyl chitosan and the oxidized sodium alginate are mixed in equal volume according to the mass ratio of 8.
8. The method for preparing a 4D-printed chitosan-based cell carrier according to claim 1, wherein the pore size of the hydroxyethyl chitosan temperature-sensitive gel involved in the step (4) is 50-100 μm;4D printing chitosan-based cell carriers, and storing the chitosan-based cell carriers in a sterile environment at the temperature of 4-15 ℃ for 6-12 months.
9. The preparation method of the 4D printing chitosan-based cell carrier according to any one of claims 1 to 8, wherein the 4D printing chitosan-based cell carrier is applied to the surface of diabetic ulcer skin after being coated with adipose-derived mesenchymal stem cells, and the wound surface is repaired by releasing the adipose-derived mesenchymal stem cells; can also be used for repairing skin burn and corneal and cervical injury.
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CN104984402A (en) * | 2015-07-14 | 2015-10-21 | 青岛大学 | Preparation method for hydroxyethyl chitosan in-situ hydrogel |
CN109749025A (en) * | 2019-02-26 | 2019-05-14 | 哈尔滨工业大学 | A kind of 4D prints preparation and the Method of printing of self-healing hydrogel material |
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