CN109503863A - A kind of injection aquagel and its preparation method and application - Google Patents
A kind of injection aquagel and its preparation method and application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
- C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
- C08J3/075—Macromolecular gels
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
- A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
- C08J2305/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2405/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2401/00 or C08J2403/00
- C08J2405/02—Dextran; Derivatives thereof
Abstract
The invention discloses a kind of injection aquagels, and preparing raw material includes chitosan, sodium β-glycerophosphate and glucan.The invention also discloses the preparation method of injection aquagel, includes the following steps: that (1) chitosan is added in HCL solution and sufficiently dissolve, chitosan solution is made；(2) glucan is added in the chitosan solution of step (1) preparation, is sufficiently dissolved, the composite solution of glucan and chitosan is made；(3) sodium β-glycerophosphate aqueous solution is provided, is then added dropwise in the composite solution of step (2), is sufficiently dissolved, the complex sol of chitosan, sodium β-glycerophosphate and glucan is made；(4) complex sol of step (3) is placed in water bath with thermostatic control and carries out gel reaction, be made chitosan, sodium β-glycerophosphate and glucan composite hydrogel to get.Hydrogel of the invention has excellent biocompatibility, is raised to 37 DEG C, and hydrogel can plastic be in situ then solid-state, have good mechanical property.
Description
Technical field
The present invention relates to technical field of biological materials, especially a kind of injection aquagel and its preparation method and application.
Background technique
MI (myocardial infarction) is a kind of acute and serious heart state, and the origin cause of formation is that the blood circulation of part cardiac muscle is unexpected
It interrupts, the damage that cardiac muscle is generated because being unable to get enough oxygen.At present when treating myocardial infarction using it is more be poly- (N-
N-isopropylacrylamide) (PNIPAM) and the modified injection aquagel formed on this basis.Hydrogel be have it is netted
Structure using water as the gel of decentralized medium, had a wide range of applications in field of biomedicine, and had lot of documents report
Prove that hydrogel is highly suitable for the reparation of myocardial infarction.Current study show that applying the injectable water-setting in myocardial infarction
Glue has poly(N-isopropylacrylamide), hyaluronic acid, polyester etc..Since hydrogel can provide the environment of similar cell epimatrix
And structure, good biocompatibility are often used as the carrier of cell, support and nutrition are provided for cell growth, in organizational project
It is widely used.
Stem cell has the function of self-replacation and differentiation, thus often combines with biomaterial, repairs to lesion
It is multiple, so that cytothesis.Stem cell is developed so far existing decades, in the injury repair of the multiple tissues of human body have research and
Using having BMSCs (mesenchymal stem cell), BADSCs (brown fat Derived Stem Cells), hCSCs in myocardial repair
(human heart stem cell) etc..Stem cell is the important composition in organizational project and regenerative medicine.Quotient is blue and green【1】It is transparent Deng proposition
Matter acid hydrogel, which wraps up mesenchymal stem cell, improves the technical solution of heart function after rat myocardial infarction model, wherein hyalomitome
Acid forms injection aquagel by chemical crosslinking, then is retained in heart injury position by the viscosity of itself, fills between marrow
Proliferation and differentiation occur for matter stem cell, new cardiac muscle cell are generated, so as to improve the heart function after myocardial infarction.
However, the plastic mechanism multiplicity of injection aquagel, forms gel by chemical crosslinking, needs to use crosslinking agent,
Crosslinking agent has certain toxicity, to can have an impact to the biocompatibility of gel, often biocompatibility is inadequate for hydrogel
It is excellent；Physical gel relies on hydrogen bond when being formed, and hydrophilic and hydrophobic, electrostatic force etc., these active forces are weaker, pass through physical mechanism shape
At gel then usual mechanical property is poor, mismatched with disease damage tissue, or there is a problem of stability difference, long-term place can
It can the spontaneous plastic of meeting；There are the acquisition of cell difficulty, difficult mass propgation, stem cells to need to keep dry during the cultivation process for stem cells technology
Property, it is more demanding to growing environment the disadvantages of.
Bibliography:
1, the hyaluronic acid gel such as Shang Qingqing, Zhou Jianye, Li Kai, which wraps up mesenchymal stem cell, improves rat heart muscle
Heart function [J] China Tissue Engineering Study, 2018,22 (5): 675-679 after infarct.
Summary of the invention
Based on the above issues, it is an object of the invention to overcome in place of above-mentioned the deficiencies in the prior art and providing one kind can infuse
Jetting gel, the hydrogel have excellent biocompatibility, and it is temperature sensitive that hydrophobic bond, hydrogen bond and electrostatic force therein have gel
Property, it is at room temperature liquid, can be mixed with UCMSCs, is raised to 37 DEG C to temperature, hydrogel can plastic be in situ then solid-state, at
The glue time is short, has good mechanical property, it is a kind of pharmaceutical carrier with great potential that preparation is simple.
To achieve the above object, the technical solution adopted by the present invention is as follows:
In the first aspect, the present invention provides a kind of injection aquagels, and the raw material for preparing of the hydrogel includes shell
Glycan, sodium β-glycerophosphate and glucan.It should be noted that injectable thermosensitive hydrogel of the invention is liquid at room temperature
Hydrogel can be injected directly into human body by syringe needle, and at 37 DEG C, it is a kind of ideal that hydrogel original position, which forms solid gel-,
Tissue engineering material has good mechanical property, easy to operate, while reducing operation damage caused by patient.
Preferably, the mass ratio of the chitosan, sodium β-glycerophosphate and glucan is 4:10:1~4.
Preferably, the raw material further includes hydrochloric acid.
Preferably, the mass ratio of the hydrochloric acid and chitosan is 1:6.
In the second aspect, the present invention provides the preparation method of above-mentioned hydrogel, include the following steps:
(1) chitosan is added in HCL solution and is sufficiently dissolved, chitosan solution is made；
(2) glucan is added in the chitosan solution of step (1) preparation, is sufficiently dissolved, glucan and chitosan is made
Composite solution；
(3) sodium β-glycerophosphate aqueous solution is provided, is then added dropwise in the composite solution of step (2), is sufficiently dissolved, system
Obtain the complex sol of chitosan, sodium β-glycerophosphate and glucan；
(4) complex sol of step (3) is placed in water bath with thermostatic control and carries out gel reaction, chitosan, β-glycerol phosphorus is made
Sour sodium and glucan composite hydrogel to get.It should be noted that the CS/DEX/ β-GP (chitosan/Portugal being obtained by this method
Glycan/sodium β-glycerophosphate) injectable thermosensitive hydrogel mechanical property with it is more excellent on biology performance, be suitable for cardiac muscle
Injury repair after infraction.
Preferably, the mass ratio of chitosan, sodium β-glycerophosphate and glucan is 4 in the complex sol of the step (3):
10:1~4.Wherein, present inventor has found through test of many times, when chitosan, β-glycerol in composite hydrogel of the invention
When the mass concentration of sodium phosphate and glucan is 0.1~0.5mg/ml, with the cell of composite hydrogel co-cultivation of the invention
Survival rate has been more than 100%.
Preferably, the mass ratio of hydrochloric acid and chitosan is 1:6 in the step (1).
Preferably, the temperature of water bath with thermostatic control is 37 DEG C in the step (4).
In the third aspect, the present invention provides above-mentioned hydrogels in preparation treatment myocardial infarction or repairs the drug of cardiac muscle
In application.
In the fourth aspect, the present invention provides a kind of drug for treating myocardial infarction, containing having the right to want in the drug
The umbilical cord mesenchymal stem cells (UCMSCs) seeking the hydrogel of first aspect and being loaded by the hydrogel.It needs to illustrate
It is that the stem cell that uses is UCMSCs in the present invention, and the prior art generally uses BMSCs (mesenchymal stem cell), but
UCMSCs derives from umbilical cord tissue, reduces the risk polluted by pathogeny, and cell viability is secure；Next stem cell content is very
It is abundant, and proliferative capacity is strong, it is better than BMSCs, immunogenicity is easy and to puerpera, baby without any damage lower than BMSCs acquisition,
No ethics dispute；UCMSCs Proliferation, Differentiation ability is very strong, has wide potential applicability in clinical practice in terms of organizational project, can
Heart injury reparation applied to myocardial infarction.
In conclusion the invention has the benefit that
Hydrogel material of the invention has syringeability, and chitosan and glucan make it have excellent bio-compatible
Property, hydrophobic bond, hydrogen bond and electrostatic force make gel have Thermo-sensitive, can mix with UCMSCs for liquid, be raised at room temperature to temperature
37 DEG C, then can be using gel in-situ as solid-state, gelation time is shorter (only needing 3min), and it is the medicine having great potential that preparation is simple
Object carrier.
Detailed description of the invention
Fig. 1 is hydrogel test flow chart of the invention；
Fig. 2 is liquid-Solid State Transformation schematic diagram of hydrogel；
Fig. 3 is the FTIR spectrum figure of each step product；
Fig. 4 is CS/2.0Dex/ β-GP rheogram (temperature-modulus)；
Fig. 5 is the scanning electron microscope (SEM) photograph for loading the hydrogel scaffold of UCMSCs；
Fig. 6 is the statistical results chart of cell activity after the composite hydrogel of various concentration is co-cultured with cell.
Specific embodiment
The present invention relates to organizational project and regenerative medicine field, present inventor utilizes chitosan, glucan and
Sodium β-glycerophosphate develops a kind of new injectable hydrogel, is a kind of injectable responsive to temperature type natural biologic material, bears
UCMSCs (umbilical cord mesenchymal stem cells) are carried to repair the heart injury after myocardial infarction.By hydrogel package stem cell note
It injects in vivo, due to the characteristic of stem cell, the cardiac muscular tissue of damage can be repaired and be regenerated.Hydrogel of the invention uses
Chitosan, and dextran modification is added, by physical crosslinking gained, relative to PNIPAM, biocompatibility is more excellent, and
Preparation process is simple, is suitable for Mirae Corp.ization and develops.
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with the drawings and specific embodiments pair
The present invention is described further.Unless otherwise instructed, reagent concentration is mass concentration in the present invention.
Embodiment 1
The preparation of injectable thermosensitive hydrogel CS/ β-GP/DEX (chitosan/sodium β-glycerophosphate/glucan) of the invention
A kind of embodiment of method, includes the following steps:
1) dilution experiment room concentrated hydrochloric acid configures the HCL solution of enough 0.1mol/L；
2) precise 400mg chitosan is dissolved in the 0.1mol/L hydrochloric acid of 18ml, and stirring and dissolving is overnight, and it is poly- to prepare shell
Sugar juice；
3) precise 500mg β-GP is dissolved in and is added in a certain amount of deionized water, and stirring concussion is sufficiently dissolved, is added
Suitable deionized water is settled to 1ml, is prepared into β-GP aqueous solution, stands 1h at 4 DEG C；
4) it is dissolved in the CS solution of 1.8ml by precise 10mg, 20mg, 30mg, 40mg glucan respectively, stirring
It is placed in 1h at 4 DEG C after completely dissolution；
5) draw 0.2ml 3) in GP solution, be slowly added into CS/DEX composite solution, while drop while stir,
0.5W/V%, 1.0W/V%, 1.5W/V%, the CS/DEX/ β-GP complex sol of 2.0W/V% is made in stirring 10 minutes respectively；
6) CS/DEX/ β-GP complex sol is placed in 37 DEG C of waters bath with thermostatic control and carries out gel reaction by, and CS/DEX/ β-is made
GP composite hydrogel.
The application of hydrogel made from 2 embodiment 1 of embodiment
(1) extraction and culture of UCMSCs (umbilical cord mesenchymal stem cells)
1) it takes health full term umbilical cord blood: being handled in 6h after umbilical cord acquisition, cut off double-sided tape catcher mark and silt
The part of blood, with sufficiently rinsing umbilical cord periphery and umbilical vein inner cavity containing dual anti-PBS buffer solution；
2) it plants block method culture UCMSCs: longitudinally cutting off blood vessel along umbilical vein inner cavity, remove umbilical vein inner membrance, residue is organized
It is cut into 3.0~5.0mm3Fritter, being affixed in advance (is 2% fetal calf serum, 40% containing volume fraction with 1mL complete medium
MCDB201,10 μ g/L platelet derived growth factors, 10 μ g/L basic fibroblast growth factors, 10 μ g/L epidermal growths
The DF12 culture medium of the factor) wetting T25 culture bottle bottom wall, bottom wall upward, be placed in 37 DEG C, volume fraction be 5% CO2Saturation
In humidified incubator, overturn after staying overnight；
3) per the above-mentioned culture medium of 1mL is added for 24 hours, 72h full dose is changed liquid, is changed weekly later liquid 2 times.It observes adherent around paste block
Cell climbs out of situation, removes paste block after 2 weeks；
4) in the 14th day colonies number of culture, more than 50 cells are calculated as colony.When cell melts up to 70%~80%
When conjunction, after the digestive juice digestion of the 1g/L ethylenediamine tetra-acetic acid of trypsase containing 1.25g/L, with (2.5~5.0) × 103/cm2It is close
Degree inoculation secondary culture, is calculated as 1st generation (P1) cell；
5) continue to cultivate with above-mentioned complete medium, the cell after passage is denoted as P2, P3, P4 ...
(2) building and zoopery of animal model
1) standby as treatment group after mixing the hydrogel to sterilize in advance (hydrogel prepared by embodiment 1) with UCMSCs
With；
2) 4 week old male Wistar rat 70, quality 180-220g.Animal is randomly divided into 5 groups, sham-operation group (n=
10), PBS group (n=15), PBS+UCMSCs group (n=15), hydrogel group (n=15), hydrogel+UCMSCs group (n=15)；
3) after yellow Jackets (20mg/kg) anesthetized rat of intraperitoneal injection 2%, preserved skin, trachea cannula, center is opened
Chest, after exposing heart, PBS group, PBS+UCMSCs group, hydrogel group, hydrogel+UCMSCs group are in left anterior descending branch proximal end 5-0
Hurtless measure silk thread is threaded and is ligatured, and sham-operation group is only threaded and do not ligatured；
4) specific as follows by 75 μ L liquid point, 3 injection infarcted myocardium boundaries after modeling success:
PBS group injects 75 μ L PBS；
PBS+UCMSCs group injects the PBS that 75 μ L contain UCMSCs；
Hydrogel group injects 75 μ L hydrogels；
Hydrogel+UCMSCs group 75 μ L UCMSCs of injection and hydrogel mixed liquor.
Then the layer-by-layer suture wall of the chest, negative pressure ventilation close thoracic cavity, after animal restores autonomous respiration, extract intubation, stop exhaling
Suction machine.Intramuscular injection 50 × 104U/d penicillin 3d is to prevent to infect；
5) the parameter characterizations hearts such as echocardiogram, electrocardiogram, LVEF (Left Ventricular Ejection Fraction) and LVFS (left room shortening rate)
Flesh recovery situation；
6) immunohistochemistry, the differentiation of the Experimental Characterizations such as histotomy UCMSCs, myocardial infarction area area etc..
Hydrogel performance test made from 3 embodiment 1 of embodiment
Test content is as shown in flow chart 1.
(1) Fourier infrared spectrum is tested
FTIR Fourier infrared spectrum is a kind of spectrum for showing molecular vibration, can identify the functional group in test substance,
Product and raw material that each step obtains are done into infrared test, whether provable target product is successfully synthesized.
Appropriate test substance (i.e. hydrogel made from embodiment 1, the CS/DEX/ β-GP of 2.0W/V%) and potassium bromide are taken,
Tabletting is ground, it is as shown in Figure 3 to obtain result for spectrometer test analysis.CS curve: 3000cm-1-3500cm-1For the flexible vibration of O-H
Dynamic absorption band, amino is in 3400cm-1-3500cm-1Region extension and shrinking vibration, 2872cm-1The respectively flexible vibration of aliphatic C-H
Dynamic absorption peak；1674cm-1For C=O stretching vibration absworption peak；1604cm-1For N-H deformation vibration the absworption peak；1383cm-1For C-
The symmetrical deformation vibration absorption peak of CH3；1322cm-1For the stretching vibration absworption peak of C-N；1251cm-1It is stretched for C-O on hexatomic ring
Contracting vibration absorption peak: 1086cm-1For the stretching vibration absworption peak of C-O.β-GP:3365cm-1For O-H stretching vibration peak, 1080cm-1Place is phosphate groups asymmetric stretching vibration absorption peak；968cm-1Place is the symmetrical stretching vibration of phosphate radical.DEX:O-H's
Stretching vibration corresponds to 3340cm-1The stretching vibration of the absorption peak at place, c h bond makes 2922cm-1Place generates characteristic absorption peak,
1641cm-1It is the C=O key of-CHO in sample that the absorption peak at place is corresponding, this at three characteristic peak illustrate that DEX has glucide
Characteristic absorption peak.
CS/DEX/ β-GP: after DEX is added, red shift occurs for O-H and N-H absorption peak, illustrates to form hydrogen bond with glucan.C-H
The bending vibration 1446cm of key-1Reduce to 1427cm-1, illustrate that chitosan and dextran molecule chain form hydrogen bond.In addition, CS/
DEX/ β-GP is in 1000-1500cm-1Absorption peak it is more wider, it may be possible between DEX and CS and β-GP generate interaction.
(2) rotational rheometer can characterize product modulus, and G' characterizes the elasticity modulus of sample, and G " characterizes the sticky mould of sample
Amount.As G' > G " when, it is believed that gel-forming, from Fig. 4 it can be seen that at 37 DEG C, CS/2.0Dex/ β-GP forms gel；From Fig. 2
As can be seen that [wherein, CS is 2.0 (W/V) % to CS/2.0Dex/ β-GP, and GP is 5.0 (W/V) %, Portugal due to the variation of temperature
Glycan Dex is 2.0 (W/V) %] hydrogel from liquid is changed into solid-state.
(3) stem cell (UCMSCs) meeting tactophily on bracket (i.e. hydrogel made from embodiment 1), utilizes Flied emission
(Fig. 5) is imaged in electronic scanner microscope, it can be seen that the pattern of cell and bracket (i.e. hydrogel).
(4) proliferation and toxicity detection of hydrogel of the invention to cell
Detection method: cell and material (hydrogel made from the method using embodiment 1) are co-cultured together, are used
Cell Counting Kit-8 (abbreviation CCK-8) reagent analysis cell Proliferation and toxicity.Specific step includes:
1) it takes health full term umbilical cord blood: being handled in 6h after umbilical cord acquisition, cut off double-sided tape catcher mark and silt
The part of blood, with sufficiently rinsing umbilical cord periphery and umbilical vein inner cavity containing dual anti-PBS buffer solution.
2) it utilizes plant block method culture UCMSCs: longitudinally cutting off blood vessel along umbilical vein inner cavity, remove umbilical vein inner membrance, it will be remaining
Tissue is cut into 3.0~5.0mm3Fritter, being affixed in advance (is 2% fetal calf serum, 40% containing volume fraction with 1mL complete medium
MCDB201,10 μ g/L platelet derived growth factors, 10 μ g/L basic fibroblast growth factors, 10 μ g/L epidermal growths
The DF12 culture medium of the factor) wetting T25 culture bottle bottom wall, bottom wall upward, be placed in 37 DEG C, volume fraction be 5% CO2Saturation
In humidified incubator, overturn after staying overnight.Per the above-mentioned culture medium of 1mL is added for 24 hours, 72h full dose is changed liquid, is changed weekly later liquid 2 times.
3) it observes attached cell around paste block and climbs out of situation, remove paste block after 2 weeks.In the 14th day colonies number of culture,
More than 50 cells are calculated as colony.When cell is merged up to 70%~80%, with the 1g/L ethylenediamine tetraacetic of trypsase containing 1.25g/L
After the digestive juice digestion of acetic acid, with (2.5~5.0) × 103/cm2Density is inoculated with secondary culture, is calculated as 1st generation (P1) cell.With
Above-mentioned complete medium continues to cultivate, and the cell after passage is denoted as P2, P3, P4.
Experimental result is as shown in fig. 6, the survival rate of cell (UCMSCs) illustrates this hair 90% or more as can be seen from Figure
The good biocompatibility of bright hydrogel material does not have toxicity to cell (UCMSCs).
Finally it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should be managed
Solution, can with modification or equivalent replacement of the technical solution of the present invention are made, without departing from technical solution of the present invention essence and
Range.
Claims (10)
1. a kind of injection aquagel, which is characterized in that the raw material for preparing of the hydrogel includes chitosan, sodium β-glycerophosphate
And glucan.
2. hydrogel according to claim 1, which is characterized in that the chitosan, sodium β-glycerophosphate and glucan
Mass ratio is 4:10:1~4.
3. hydrogel according to claim 1, which is characterized in that the raw material further includes hydrochloric acid.
4. hydrogel according to claim 3, which is characterized in that the mass ratio of the hydrochloric acid and chitosan is 1:6.
5. the preparation method of the described in any item hydrogels of Claims 1 to 4, which comprises the steps of:
(1) chitosan is added in HCL solution and is sufficiently dissolved, chitosan solution is made；
(2) glucan is added in the chitosan solution of step (1) preparation, is sufficiently dissolved, answering for glucan and chitosan is made
Close solution；
(3) sodium β-glycerophosphate aqueous solution is provided, is then added dropwise in the composite solution of step (2), is sufficiently dissolved, shell is made
The complex sol of glycan, sodium β-glycerophosphate and glucan；
(4) complex sol of step (3) is placed in water bath with thermostatic control and carries out gel reaction, chitosan, sodium β-glycerophosphate is made
With glucan composite hydrogel to get.
6. preparation method according to claim 5, which is characterized in that chitosan, β-in the complex sol of the step (3)
The mass ratio of sodium glycero-phosphate and glucan is 4:10:1~4.
7. preparation method according to claim 5, which is characterized in that the quality of hydrochloric acid and chitosan in the step (1)
Than for 1:6.
8. preparation method according to claim 5, which is characterized in that the temperature of water bath with thermostatic control is 37 in the step (4)
℃。
9. the described in any item hydrogels of Claims 1 to 4 answering in preparation treatment myocardial infarction or the drug for repairing cardiac muscle
With.
10. a kind of drug for treating myocardial infarction, which is characterized in that contain any one of Claims 1 to 4 institute in the drug
The hydrogel stated and the umbilical cord mesenchymal stem cells loaded by the hydrogel.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022143244A1 (en) * | 2020-12-30 | 2022-07-07 | 江南大学 | Preparation method for cross-linked hydrogel for muscle stem cell culture, and application thereof |
| CN116173307A (en) * | 2023-01-31 | 2023-05-30 | 海南医学院 | Preparation method and application of citric acid chitosan hydrogel |
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| US20220315711A1 (en) * | 2020-12-30 | 2022-10-06 | Jiangnan University | Preparation Method and Use of Crosslinked Hydrogel for Muscle Stem Cell Culture |
| US11629236B2 (en) * | 2020-12-30 | 2023-04-18 | Jiangnan University | Preparation method and use of crosslinked hydrogel for muscle stem cell culture |
| CN116173307A (en) * | 2023-01-31 | 2023-05-30 | 海南医学院 | Preparation method and application of citric acid chitosan hydrogel |
| CN116173307B (en) * | 2023-01-31 | 2023-10-24 | 海南医学院 | Preparation method and application of citric acid chitosan hydrogel |
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