CN111704729A - 一种基于天然高分子且强度和成胶时间可调的水凝胶细胞支架 - Google Patents
一种基于天然高分子且强度和成胶时间可调的水凝胶细胞支架 Download PDFInfo
- Publication number
- CN111704729A CN111704729A CN202010627869.4A CN202010627869A CN111704729A CN 111704729 A CN111704729 A CN 111704729A CN 202010627869 A CN202010627869 A CN 202010627869A CN 111704729 A CN111704729 A CN 111704729A
- Authority
- CN
- China
- Prior art keywords
- gelatin
- sodium alginate
- carboxymethyl chitosan
- solution
- oxidized
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000000017 hydrogel Substances 0.000 title claims abstract description 53
- 229920005615 natural polymer Polymers 0.000 title description 8
- 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 abstract description 90
- 108010010803 Gelatin Proteins 0.000 claims abstract description 90
- 229920000159 gelatin Polymers 0.000 claims abstract description 90
- 239000008273 gelatin Substances 0.000 claims abstract description 90
- 235000019322 gelatine Nutrition 0.000 claims abstract description 90
- 235000011852 gelatine desserts Nutrition 0.000 claims abstract description 90
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 90
- 239000000661 sodium alginate Substances 0.000 claims abstract description 90
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 90
- 229920001661 Chitosan Polymers 0.000 claims abstract description 78
- 239000000243 solution Substances 0.000 claims abstract description 60
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 claims abstract description 53
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 claims abstract description 32
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 26
- 238000002156 mixing Methods 0.000 claims abstract description 20
- 125000003172 aldehyde group Chemical group 0.000 claims abstract description 19
- 239000007864 aqueous solution Substances 0.000 claims abstract description 10
- 230000001590 oxidative effect Effects 0.000 claims abstract description 4
- 239000011259 mixed solution Substances 0.000 claims abstract 2
- 239000000499 gel Substances 0.000 claims description 36
- 125000003277 amino group Chemical group 0.000 claims description 30
- 238000006243 chemical reaction Methods 0.000 claims description 17
- 238000007254 oxidation reaction Methods 0.000 claims description 17
- 230000003647 oxidation Effects 0.000 claims description 16
- 238000003860 storage Methods 0.000 claims description 15
- WTDHULULXKLSOZ-UHFFFAOYSA-N Hydroxylamine hydrochloride Chemical compound Cl.ON WTDHULULXKLSOZ-UHFFFAOYSA-N 0.000 claims description 8
- 239000002904 solvent Substances 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 230000015572 biosynthetic process Effects 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 2
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 25
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 15
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 8
- 239000000463 material Substances 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 230000001276 controlling effect Effects 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 235000019766 L-Lysine Nutrition 0.000 description 4
- 239000004472 Lysine Substances 0.000 description 4
- -1 cells Substances 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- 238000000862 absorption spectrum Methods 0.000 description 3
- 239000003431 cross linking reagent Substances 0.000 description 3
- 125000000524 functional group Chemical group 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- IYMAXBFPHPZYIK-BQBZGAKWSA-N Arg-Gly-Asp Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O IYMAXBFPHPZYIK-BQBZGAKWSA-N 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 2
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 2
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 229920001436 collagen Polymers 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 210000002744 extracellular matrix Anatomy 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- 238000000954 titration curve Methods 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- NHJVRSWLHSJWIN-UHFFFAOYSA-N 2,4,6-trinitrobenzenesulfonic acid Chemical compound OS(=O)(=O)C1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O NHJVRSWLHSJWIN-UHFFFAOYSA-N 0.000 description 1
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 1
- 150000008545 L-lysines Chemical class 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 239000002262 Schiff base Substances 0.000 description 1
- 150000004753 Schiff bases Chemical class 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 108010072041 arginyl-glycyl-aspartic acid Proteins 0.000 description 1
- 239000013060 biological fluid Substances 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 229940045110 chitosan Drugs 0.000 description 1
- 229960005188 collagen Drugs 0.000 description 1
- 210000004292 cytoskeleton Anatomy 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000004146 energy storage Methods 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 229940014259 gelatin Drugs 0.000 description 1
- 238000001879 gelation Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 229920001477 hydrophilic polymer Polymers 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000000518 rheometry Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 230000009772 tissue formation Effects 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Images
Classifications
-
- 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
-
- 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
- C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
- C08J3/246—Intercrosslinking of at least two polymers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0068—General culture methods using substrates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
-
- 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
- C08J2389/00—Characterised by the use of proteins; Derivatives thereof
-
- 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
- C08J2405/04—Alginic acid; Derivatives thereof
-
- 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
- C08J2405/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/54—Collagen; Gelatin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/70—Polysaccharides
- C12N2533/72—Chitin, chitosan
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/70—Polysaccharides
- C12N2533/74—Alginate
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Dispersion Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Oncology (AREA)
- Reproductive Health (AREA)
- Materials For Medical Uses (AREA)
- Medicinal Preparation (AREA)
Abstract
本申请提供了一种制备强度和成胶时间可调的明胶‑壳聚糖水凝胶细胞支架的方法及通过该方法制备的水凝胶细胞支架。所述方法包括如下步骤:(1)制备氧化海藻酸钠:使海藻酸钠与高碘酸钠发生氧化反应得到部分链段含醛基的氧化海藻酸钠;(2)测定步骤(1)所得氧化海藻酸钠的醛基含量;(3)检测明胶中的氨基含量;(4)分别配制浓度为15wt%的明胶溶液和浓度为4wt%的羧甲基壳聚糖溶液;(5)将步骤(4)所得的明胶水溶液和羧甲基壳聚糖水溶液混合;(6)在所述明胶和羧甲基壳聚糖中总氨基量与所述氧化海藻酸钠中醛基量等摩尔的条件下,使步骤(5)所得明胶水溶液和羧甲基壳聚糖水溶液的混合溶液与步骤(1)中所得氧化海藻酸钠混合反应。
Description
技术领域
本申请涉及天然高分子水凝胶领域,具体而言,涉及一种制备强度和成胶时间可调的明胶-壳聚糖水凝胶细胞支架的方法。
背景技术
水凝胶是指三维交联的亲水性聚合物网络,可以吸收和保留大量的水或生物液体。水凝胶的几个独特的特征,主要包括类似组织的细胞外基质(ECM),支持细胞的增殖和迁移,控制生长因子的释放,对周围组织的机械刺激最小,支持细胞生长和增殖的营养扩散等。这些特性使其可以作为生长因子、细胞、药物和基因的载体,使其在组织工程和再生医学中获得了广泛的应用[1-5]。其中,天然高分子水凝胶是作为最重要的组织工程支架材料之一,为细胞在支架材料中的生长和分化,组织形成,更好的调节器官和细胞之间的相互作用等提供了理想的环境,同时具有更好的生物相容性,较低的毒性等优点[2,6]。另外,大多数天然聚合物都具有易于与配体结合、容易进行交联以及其他修饰的反应位点,从而为构建更加适合多种临床应用的生物材料提供了可能。总的来说,天然聚合物主要有以下几类,蛋白质类包括胶原、明胶、纤维蛋白、溶菌酶等,多糖类如透明质酸、海藻酸盐、壳聚糖、琼脂糖等,核酸类等。基于这些天然高分子材料构建的水凝胶获得了或得广泛的关注和应用开发。但是进一步构建性能更加优异,结构具有更易于调节的天然高分子水凝胶材料依然是该领域的重要挑战之一。
壳聚糖具有良好的生物相容性、无毒性、低成本等优点,被广泛应用于药物制剂、食品添加剂和组织工程中[7]。明胶是由胶原蛋白部分水解而成的产物,其生物相容性和生物降解性是生物医学工程研究的热点,其含有的Arg-Gly-Asp(RGD)样序列,可以促进细胞粘附和迁移。因此将明胶材料与壳聚糖混合构建的水凝胶材料,可以作为优异的细胞支架材料[8-10]。以明胶-壳聚糖为原料制备的水凝胶制备方法包括利用辐射诱导交联法[11]、原位沉淀法[12],添加小分子交联剂[13]等方法。以这些方法制备的明胶-壳聚糖水凝胶分别在创伤愈合、软骨组织工程、缺血组织再生等方面显示了以很好的应用前景。但是利用这些方法构建明胶-壳聚糖凝胶材料,依然存在一些问题,制约了其更加广泛的应用,如溶解壳聚糖需要添加醋酸后期需要将醋酸中和,制备凝胶过程需要一系列繁琐耗时的试验步骤,不能保证所有反应官能团参与反应即可能存在未反应的官能团对细胞产生毒性,制备过程引入一些交联试剂也不可避免的会对细胞产生一定的毒性等。因此,急需进一步开发新型的构建明胶-壳聚糖水凝胶的方法。
为了解决以上问题,在该项目中,我们首先制备了含有醛基的天然高分子氧化海藻酸钠,利用氧化海藻酸钠产生的醛基与明胶以及羧甲基壳聚糖分子链上的氨基发生席夫碱反应,构建水凝胶新型的明胶-壳聚糖水凝胶,该方法具有成胶时间可控,凝胶强度易于调节等优点,最终得到的明胶-壳聚糖水凝胶具有良好的生物相容性并易于细胞粘附,是一种理想的水凝胶细胞支架材料。
参考文献:
[1]T.Vermonden,R.Censi,W.E.Hennink,Hydrogels for Protein Delivery,Chem.Rev.112(5)(2012)2853-2888.
[2]Y.L.Li,J.Rodrigues,H.Tomas,Injectable and biodegradable hydrogels:gelation,biodegradation and biomedical applications,Chem.Soc.Rev.41(6)(2012)2193-2221.
[3]N.E.Fedorovich,J.Alblas,J.R.de Wijn,W.E.Hennink,A.J.Verbout,W.J.A.Dhert,Hydrogels as extracellular matrices for skeletal tissueengineering:state-of-the-art and novel application in organ printing,TissueEngineering 13(8)(2007)1905-1925.
[4]Y.Tabata,Tissue regeneration based on growth factor release,TissueEngineering 9(2003)S5-S15.
[5]A.Sivashanmugam,R.A.Kumar,M.V.Priya,S.V.Nair,R.Jayakumar,Anoverview of injectable polymeric hydrogels for tissue engineering,Eur.Polym.J.72(2015)543-565.
[6]P.M.Kharkar,K.L.Kiick,A.M.Kloxin,Designing degradable hydrogelsfor orthogonal control of cell microenvironments,Chem.Soc.Rev.42(17)(2013)7335-7372.
[7]M.Prabaharan,Chitosan derivatives as promising materials forcontrolled drug delivery,Journal of Biomaterials Applications 23(1)(2008)5-36.
[8]H.Shin,S.Jo,A.G.Mikos,Modulation of marrow stromal osteoblastadhesion on biomimetic oligo[poly(ethylene glycol)fumarate]hydrogels modifiedwith Arg-Gly-Asp peptides and a poly(ethylene glycol)spacer,Journal ofBiomedical Materials Research 61(2)(2002)169-179.
[9]D.J.Coletta,A.Ibanez-Fonseca,L.R.Missana,M.V.Jammal,E.J.Vitelli,M.Aimone,F.Zabalza,J.P.M.Issa,M.Alonso,J.C.Rodriguez-Cabello,S.Feldman,BoneRegeneration Mediated by a Bioactive and Biodegradable Extracellular Matrix-Like Hydrogel Based on Elastin-Like Recombinamers,Tissue Engineering Part A23(23-24)(2017)1361-1371.
[10]I.Jun,K.M.Park,D.Y.Lee,K.D.Park,H.Shin,Control of adhesion,focaladhesion assembly,and differentiation of myoblasts by enzymaticallycrosslinked cell-interactive hydrogels,Macromol.Res.19(9)(2011)911-920.
[11]C.Yang,L.Xu,Y.Zhou,X.M.Zhang,X.Huang,M.Wang,Y.Han,M.L.Zhai,S.C.Wei,J.Q.Li,A green fabrication approach of gelatin/CM-chitosan hybridhydrogel for wound healing,Carbohydr.Polym.82(4)(2010)1297-1305.
[12]Z.S.Shen,X.Cui,R.X.Hou,Q.Li,H.X.Deng,J.Fu,Tough biodegradablechitosan-gelatin hydrogels via in situ precipitation for potential cartilagetissue engineering,Rsc Advances 5(69)(2015)55640-55647.
[13]N.C.Cheng,W.J.Lin,T.Y.Ling,T.H.Young,Sustained release ofadipose-derived stem cells by thermosensitive chitosan/gelatin hydrogel fortherapeutic angiogenesis,Acta Biomaterialia 51(2017)258-267.
发明内容
虽然基于明胶-壳聚糖水凝胶有良好的生物学应用,不过壳聚糖水溶性很差、并且该类水凝胶合成过程相对繁琐耗时。为了解决上述问题,本发明提供了一种制备强度和成胶时间可调的明胶-壳聚糖水凝胶细胞支架的方法,该方法中,通过氧化海藻酸钠产生的醛基与明胶以及羧甲基壳聚糖分子链上的氨基发生反应形成交联网络。鉴于明胶分子链具有相对较低的氨基含量而羧甲基壳聚糖分子链上具有较高的氨基含量,故明胶和羧甲基壳聚糖与氧化海藻酸钠的反应速率不同,从而可以通过控制明胶和羧甲基壳聚糖的比例来调控反应时间、控制聚合物交联网络。
本发明所述的制备强度和成胶时间可调的明胶-壳聚糖水凝胶细胞支架的方法将已经商品化的分子链中氨基含量低的明胶和分子链中氨基含量高的羧甲基壳聚糖混合,并使明胶中氨基的摩尔量占明胶和羧甲基壳聚糖中总氨基摩尔量的50%或60%;再与54%氧化度(氧化程度)的海藻酸钠直接混合反应形成两种不同密度的交联网络的水凝胶,赋予了水凝胶在凝胶模量和凝胶时间具有很大范围的可调节性。氧化海藻酸钠产生的醛基与低氨基含量的明胶和高氨基含量的羧甲基壳聚糖上的氨基发生席夫碱反应,形成两种不同密度的交联网络的水凝胶。通过控制明胶中的氨基与羧甲基壳聚糖中的氨基比例,达到控制水凝胶的储能模量、凝胶时间的目的。羧甲基壳聚糖和明胶的结构以及官能团反应原理如图1。通过分别测定氧化海藻酸钠分子链的醛基含量、明胶中的氨基含量,然后定量的调节明胶与羧甲基壳聚糖的比例。
本发明所述的制备强度和成胶时间可调的明胶-壳聚糖水凝胶细胞支架的方法包括如下步骤:
(1)制备氧化海藻酸钠:首先使海藻酸钠与高碘酸钠发生氧化反应得到部分链段含醛基的氧化海藻酸钠,如
例如,该合成可参考文献Carbohydrate Research 340(2005)1425-1429。氧化海藻酸钠的FT-IR表征见图3。优选地,所述氧化海藻酸钠是在高碘酸钠与海藻酸钠分子链结构单元的摩尔比为1:1.5的条件下制备的,得到的氧化海藻酸钠的氧化度为54%;
(2)测定氧化海藻酸钠的醛基含量;优选地,通过盐酸羟胺法测定氧化海藻酸钠的醛基含量(如,具体过程见图4、图5,检测方法参考文献Biomacromolecules 2014,15,4495-4508);
(3)检测明胶中的氨基含量;优选地,通过TNBSA试剂(TNBSA法)检测明胶中的氨基含量(例如,见图6);
(4)分别配制浓度为15wt%的明胶溶液(溶剂优选为10mM PBS)和浓度为4wt%的羧甲基壳聚糖溶液(溶剂优选为10mM PBS);
(5)将上步所得的明胶水溶液和羧甲基壳聚糖水溶液混合,优选地,使明胶中氨基摩尔量占明胶和羧甲基壳聚糖中总氨基摩尔量的50%-60%;
(6)在所述明胶和羧甲基壳聚糖中总氨基量与所述氧化海藻酸钠中醛基量等摩尔的条件下,使步骤(5)溶液与步骤(1)中所得氧化海藻酸钠(可配制成溶液,溶剂优选为10mMPBS;浓度范围优选5wt%~10wt%)混合反应,从而得到具有两种不同密度的交联网络:由明胶与氧化海藻酸钠形成的低密度交联网络,和由羧甲基壳聚糖与氧化海藻酸钠形成的高密度交联网络。
本申请所述方法通过控制上述两种交联网络的含量来调节所制备的水凝胶的模量、凝胶时间。
在优选的实施方式中,步骤(1)中,所述海藻酸钠与高碘酸钠的氧化反应中,在乙醇/水1:1混合溶剂、室温条件下,海藻酸钠结构单元与高碘酸钠的摩尔比为1.5:1;
在优选的实施方式中,当在步骤(6)中采用5%氧化海藻酸钠溶液时,所述凝胶时间在1min 50s到30min之间,所述储能模量为0.75Pa到210Pa范围之间;更优选地,当明胶与羧甲基壳聚糖按氨基含量按1:1混合,凝胶时间为3min 45s,储能模量为99.9Pa;
优选地,采用10%氧化海藻酸钠溶液时,当明胶-羧甲基壳聚糖混合体系中明胶的氨基含量从50%提高到60%时,所述凝胶时间从瞬间形成凝胶到140s之间,所述储能模量为251.9Pa至57.1Pa。
有益效果
反应过程简单可控,三种天然高分子之间的相互反应,无外加交联剂,可有效避免生物毒性。
通过合理控制高碘酸钠与海藻酸钠的比例可获得一定氧化程度的海藻酸钠,得到分子链上含醛基的氧化海藻酸钠,并且可以对分子链上醛基含量定量分析(表1),本发明中控制高碘酸钠与海藻酸钠分子链结构单元的摩尔比为1:1.5,得到氧化度为54%的氧化海藻酸钠分子。对明胶分子链上的氨基进行定量分析(图6)后,可以大致得到明胶分子链中约5000分子量含有一个氨基,由于不同商品批次的明胶的氨基含量可能不同,所以通过该方法可以确定不同商品明胶的氨基含量以便进一步控制形成水凝胶中明胶的含量。
用氧化后的海藻酸钠进行成胶实验,首先通过改变海藻酸钠的浓度,利用10wt%和5wt%的海藻酸钠溶液进行成胶。对于5wt%的海藻酸钠溶液,单独使用明胶形成的凝胶,凝胶时间高达30min以上(图7a),单独使用羧甲基壳聚糖溶液凝胶时间在1min 50s就可以形成凝胶(图7b)。而将明胶与羧甲基壳聚糖按氨基含量1:1混合后凝胶时间控制为3min45s(图7c),所以通过调节明胶与羧甲基壳聚糖中的氨基比例就可以使凝胶时间在1min50s到30min之间调节,而反应达到15min时储能模量也可以控制在从很低的储能模量0.75Pa到210Pa范围之间(表3)。此外,对于10%海藻酸钠溶液形成的水凝胶,当控制明胶和羧甲基壳聚糖的氨基含量为1:1时,体系在混合后很短的时间就形成凝胶,以致于流变仪测试得不到凝胶时间,对比与5%的相同体系,储能模量从210Pa到251.9Pa(图7d、表3)。说明在各个反应分子比例相同的情况下,通过改变反应分子的浓度可以很好的控制凝胶时间和凝胶的模量。
另外,明胶对比与壳聚糖,分子链的氨基含量明显较少,明胶与氧化海藻酸钠的反应速率更高,当改变少量壳聚糖的含量即可以明显改变实验速率。当明胶-羧甲基壳聚糖混合体系中明胶的氨基含量从50%提高到60%时,体系从在很短的时间内就形成凝胶过渡到140s才形成凝胶,凝胶的储能模量从251.9Pa过渡到57.1Pa。而明胶在明胶-羧甲基壳聚糖中的质量分数只从95.8%提高到97.2%,在明胶含量变化很小的情况下,就实现了凝胶的性能调控。
另外我们通过将Hela细胞包裹在明胶与羧甲基壳聚糖氨基含量为1:1的体系与5%氧化海藻酸钠溶液形成的水凝胶中,通过FDA/PI染色可以发现Hela细胞在水凝胶的生存状态良好,很少有细胞死亡(图9),说明该水凝胶的具有很好的生物相容性。
附图说明
图1示出了羧甲基壳聚糖结构、明胶结构及凝胶中的反应机理。其中,n为羧甲基壳聚糖结构单元数;
图2示出了本发明中的海藻酸钠与高碘酸钠的反应机理。其中,m是海藻酸钠中被氧化的结构单元数,n是海藻酸钠结构单元数。
图3示出了海藻酸钠和氧化后的海藻酸钠的FTIR对比。
图4示出了盐酸羟胺滴定法原理,其中,V是消耗的NaOH体积,mL;c是NaOH的浓度,mol/L;w是氧化海藻酸钠质量,g。
图5示出了氧化海藻酸钠的盐酸羟胺-滴定曲线及滴定曲线的一次微分曲线。其中,氧化海藻酸钠(海藻酸钠结构单元与高碘酸钠摩尔比为1.5:1)0.0518g,0.25mol/LNaOH,氧化度为54%。
图6.a:L-lysine系列标准溶液紫外吸收光谱;b:不同浓度明胶溶液的紫外吸收光谱;c:L-lysine紫外吸收强度与浓度曲线及相同吸收强度对应的明胶浓度;d:实验组不同明胶浓度计算得到的分子量,可得明胶中约5000分子量含有一个氨基。
图7示出了水凝胶的流变学分析。其中,15%wt的明胶溶液和4%wt的羧甲基壳聚糖溶液,氧化度为54%的海藻酸钠,溶剂为10mM PBS。其中,a:5wt%氧化海藻酸钠溶液与明胶溶液形成的水凝胶,凝胶时间35min 10s;b:5wt%氧化海藻酸钠溶液与羧甲基壳聚糖溶液形成的水凝胶,凝胶时间1min 50s;c:5wt%氧化海藻酸钠溶液与明胶和羧甲基壳聚糖溶液(氨基含量1:1)形成的水凝胶,凝胶时间3min 45s;d:10wt%氧化海藻酸钠溶液与明胶和羧甲基壳聚糖溶液(氨基含量1:1)形成的水凝胶。
图8示出了对浓度为5%氧化海藻酸钠与占总氨基含量不同比例的明胶与羧甲基壳聚糖混合溶液反应,形成凝胶的凝胶时间和在15min时的储能模量图,随着明胶氨基占总氨基摩尔含量的增加,凝胶时间增加,凝胶模量降低。(其中,“Propotion of Amino(氨基比例)”指明胶中氨基摩尔量占明胶和羧甲基壳聚糖中总氨基摩尔量的百分比)。
图9示出了将Hela包裹在明胶与羧甲基壳聚糖中的氨基比例为1:1和5wt%氧化度为54%的海藻酸钠形成的水凝胶中,在37℃,5%CO2培养箱培养24h后,用FDA/PI试剂检测Hela细胞在水凝胶中的存活情况。白色圆圈外部表示活细胞,白色圆圈内表示死细胞。
具体实施方式
海藻酸钠的氧化、相关基团的测试、水凝胶的制备及细胞的活性测试如下:
实施例一、氧化海藻酸钠的制备
(1)在250mL圆底烧瓶中放入磁子,加入海藻酸钠(Alfa Aesar,Product NO:C832672;)(5g,25.25mmol,)和25ml乙醇,加入预先准备好的高碘酸钠(Aladdin)溶液(3.6g,16.83mmol高碘酸钠溶解于25ml去离子水中),室温下在磁力搅拌器上避光搅拌6h,。然后加入3ml乙二醇反应1h。用3500kDa透析袋透析3天后,冷冻干燥。得白色泡沫状固体3.6g,产率72%。
(2)取0.0518g的氧化海藻酸钠,溶解于20ml 0.25mol/L的盐酸羟胺溶液中,反应24h后,***pH计并用0.25mol/L NaOH溶液滴定反应溶液,pH达到4.5左右停止滴定,作pH-V(NaOH)图,进行一阶微分,即可得到实际消耗的NaOH体积,反应机理如图4。
(3)计算海藻酸钠(A)与高碘酸钠(B)反应得到被氧化的海藻酸钠结构单元生成的醛基对应的海藻酸钠分子量Malg,如表1;
表1
A结构单元(mol) | B(mol) | 氧化度% | M<sub>alg</sub> |
1.5 | 1 | 54 | 366 |
实施例二、明胶中氨基含量的测试。
(1)首先将5%TNBSA(三硝基苯磺酸,Sigma公司)溶液用0.1M NaHCO3缓冲溶液稀释500倍,现配现用。配置系列0.5、1、2、4、6、8、10、20ug/mL L-lysine标准溶液,20、100、200ug/mL明胶(Aladdin,Item NO:G108394;)溶液,溶剂为0.1M NaHCO3。分别将250uLTNBSA稀释液加入到500ul L-lysine和明胶溶液中。在37℃下反应2h。
(2)分别将250uL 10%SDS(十二烷基硫酸钠,分析纯)和125uL 1M HCL加入到每个样品中停止反应。测试每个溶液在的紫外吸收光谱,以L-lysine溶液在339nm处的吸光度对浓度做标准曲线,计算明胶中的氨基含量约为5000分子量含有一个氨基。
实施例三、水凝胶的制备及流变学测试
(1)首先用10mM PBS为溶剂配制5wt%和10wt%的氧化度为54%的氧化海藻酸钠溶液、15wt%明胶溶液、4wt%羧甲基壳聚糖(Macklin,Product No:C832672;)溶液。如表2;
表2
氧化海藻酸钠 | 明胶 | 羧甲基壳聚糖 | |
浓度(wt%) | 5(10) | 15 | 4 |
分子量 | 183 | 5000 | 218 |
比例 | 300ul(150ul) | 683ul | 112ul |
(2)在确保氨基与醛基等摩尔反应的条件下,以明胶中氨基摩尔量占明胶和羧甲基壳聚糖总氨基摩尔量的50%的比例,使明胶溶液和羧甲基壳聚糖溶液分别与10wt%和5wt%的氧化度为54%的氧化海藻酸钠溶液反应,首先用移液枪将明胶溶液与羧甲基壳聚糖溶液在5ml离心管中按照相应比例充分混合(另外两组仅加入明胶或羧甲基壳聚糖),然后用移液枪加入相应海藻酸钠溶液,立刻旋涡振荡器使其充分混合5~10s,然后立即用移液枪吸取进行流变测试。该水凝胶的储能模量和损耗模量在旋转流变仪的时间扫描模式下,如表3;
表3
G’、G”分别为水凝胶在15min的储能模量和损耗模量。“/”表示凝胶时间过快,无法通过流变仪测得G’与G”交点。
(3)在确保氨基与醛基等摩尔反应的条件下,分别以明胶中的氨基摩尔量占明胶和羧甲基壳聚糖总氨基摩尔量的50%、60%的比例,使明胶溶液和羧甲基壳聚糖溶液与10wt%的氧化度为54%的海藻酸钠溶液反应制备水凝胶,如表4;
表4;
首先用移液枪将明胶溶液与羧甲基壳聚糖溶液在5ml离心管中按照相应比例充分混合,然后用移液枪加入相应海藻酸钠,立刻旋涡振荡器使其充分混合5-10s,然后立即用移液枪吸取进行流变测试。上述该水凝胶的储能模量和损耗模量在旋转流变仪的时间扫描模式下,角频率6.28rad/s,剪切应变10%测试。结果如表5;
表5;
G’、G”分别为水凝胶在15min的储能模量和损耗模量。“/”表示凝胶时间过快,无法通过流变仪测得G’与G”交点。
实施例四、水凝胶生物相容性测试
(1)用10mM PBS配制5%氧化度为54%的海藻酸钠溶液,分别用DMEM(Hyclone)配制3mL 20wt%明胶溶液和1mL 4wt%羧甲基壳聚糖溶液。
(2)将正常培养的长满9mm培养皿Hela细胞(来源于中国科学院上海细胞库)用PBS清洗三次,用少量胰蛋白酶消化,并用1mL DMEM重新分散,将该1mL细胞溶液加入到3mL明胶溶液中,使其最终浓度为15wt%,轻轻混合均匀。取683uL含细胞的明胶溶液与112uL羧甲基壳聚糖溶液混合,再加入300uL海藻酸钠溶液,轻轻混合,加入到24孔培养皿中,在37℃培养箱培养1h后,再在凝胶表面加入500mL DMEM培养24h。
(3)24h后,用10mM PBS清洗水凝胶表面3次,随后加入200uL FDA和200uL PI溶液,培养20分钟。然后用10mM PBS清洗3次,除去多余的染色剂。用荧光显微镜观察,白色圆圈外部表示活细胞,白色圆圈内表示死细胞。见图9。通过FDA/PI染色可以发现Hela细胞在水凝胶的生存状态良好,很少有细胞死亡,说明该水凝胶具有很好的生物相容性。
Claims (7)
1.一种制备强度和成胶时间可调的明胶-壳聚糖水凝胶细胞支架的方法,该方法包括如下步骤:
(1)制备氧化海藻酸钠:使海藻酸钠与高碘酸钠发生氧化反应得到部分链段含醛基的氧化海藻酸钠;
(2)测定步骤(1)所得氧化海藻酸钠的醛基含量;
(3)检测明胶中的氨基含量;
(4)分别配制浓度为15wt%的明胶溶液和浓度为4wt%的羧甲基壳聚糖溶液;
(5)将步骤(4)所得的明胶水溶液和羧甲基壳聚糖水溶液混合;
(6)在所述明胶和羧甲基壳聚糖中总氨基量与所述氧化海藻酸钠中醛基量等摩尔的条件下,使步骤(5)所得明胶水溶液和羧甲基壳聚糖水溶液的混合溶液与步骤(1)中所得氧化海藻酸钠混合反应,从而得到具有两种不同密度的交联网络:由明胶与氧化海藻酸钠形成的低密度交联网络,和由羧甲基壳聚糖与氧化海藻酸钠形成的高密度交联网络。
2.根据权利要求1所述的方法,其中,在步骤(1)中,所述氧化海藻酸钠是在高碘酸钠与海藻酸钠分子链结构单元的摩尔比为1:1.5的条件下制备的,得到的氧化海藻酸钠的氧化度为54%;
优选地,在步骤(1)中,所述海藻酸钠与高碘酸钠的氧化反应中,在乙醇/水1:1混合溶剂、室温条件下,海藻酸钠结构单元与高碘酸钠的摩尔比为1.5:1。
3.根据权利要求1所述的方法,其中,在步骤(2)中,通过盐酸羟胺法测定氧化海藻酸钠的醛基含量。
4.根据权利要求1所述的方法,其中,在步骤(3)中,通过TNBSA试剂检测明胶中的氨基含量。
5.根据权利要求1所述的方法,其中,在步骤(5)中,使明胶中氨基摩尔量占明胶和羧甲基壳聚糖中总氨基摩尔量的50%-60%。
6.根据权利要求1所述的方法,其中,在步骤(6)中,当在步骤(6)中采用5%氧化海藻酸钠溶液时,所述凝胶时间在1min 50s到30min之间,所述储能模量为0.75Pa到210Pa范围之间;更优选地,当明胶与羧甲基壳聚糖按氨基含量按1:1混合,凝胶时间为3min45s,储能模量为99.9Pa;
当采用10%氧化海藻酸钠溶液时,当明胶-羧甲基壳聚糖混合体系中明胶的氨基含量从50%提高到60%时,所述凝胶时间从瞬间形成凝胶到140s之间,所述储能模量为251.9Pa至57.1Pa。
7.根据权利要求1至6中任意一项所述的方法制备的强度和成胶时间可调的明胶-壳聚糖水凝胶细胞支架。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010627869.4A CN111704729B (zh) | 2020-07-02 | 2020-07-02 | 一种基于天然高分子且强度和成胶时间可调的水凝胶细胞支架 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010627869.4A CN111704729B (zh) | 2020-07-02 | 2020-07-02 | 一种基于天然高分子且强度和成胶时间可调的水凝胶细胞支架 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN111704729A true CN111704729A (zh) | 2020-09-25 |
CN111704729B CN111704729B (zh) | 2022-05-10 |
Family
ID=72545856
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010627869.4A Active CN111704729B (zh) | 2020-07-02 | 2020-07-02 | 一种基于天然高分子且强度和成胶时间可调的水凝胶细胞支架 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN111704729B (zh) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113318276A (zh) * | 2021-03-29 | 2021-08-31 | 中山大学附属第一医院 | 一种多重交联可注射水凝胶的制备方法及其制备方法和应用 |
CN115887745A (zh) * | 2022-11-23 | 2023-04-04 | 北京航空航天大学 | 一种止血水凝胶及其制备方法和应用 |
CN115920120A (zh) * | 2022-12-01 | 2023-04-07 | 江南大学附属医院 | 促进伤口再生性愈合的水凝胶复合物及其制备方法 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104479150A (zh) * | 2014-10-29 | 2015-04-01 | 上海大学 | 多重交联多糖可注射型水凝胶制备方法 |
CN107149700A (zh) * | 2017-05-22 | 2017-09-12 | 浙江大学 | 一种三组份生物胶水及其制备与应用 |
CN110201219A (zh) * | 2019-05-14 | 2019-09-06 | 浙江理工大学 | 一种可注射且快速凝胶化的复合水凝胶及其制备方法 |
CN110917385A (zh) * | 2019-11-20 | 2020-03-27 | 山东百多安医疗器械有限公司 | 一种自修复快速封合医用胶及其制备方法 |
CN112999418A (zh) * | 2021-03-02 | 2021-06-22 | 华东数字医学工程研究院 | 医用水凝胶组合物、医用水凝胶及其制备方法 |
-
2020
- 2020-07-02 CN CN202010627869.4A patent/CN111704729B/zh active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104479150A (zh) * | 2014-10-29 | 2015-04-01 | 上海大学 | 多重交联多糖可注射型水凝胶制备方法 |
CN107149700A (zh) * | 2017-05-22 | 2017-09-12 | 浙江大学 | 一种三组份生物胶水及其制备与应用 |
CN110201219A (zh) * | 2019-05-14 | 2019-09-06 | 浙江理工大学 | 一种可注射且快速凝胶化的复合水凝胶及其制备方法 |
CN110917385A (zh) * | 2019-11-20 | 2020-03-27 | 山东百多安医疗器械有限公司 | 一种自修复快速封合医用胶及其制备方法 |
CN112999418A (zh) * | 2021-03-02 | 2021-06-22 | 华东数字医学工程研究院 | 医用水凝胶组合物、医用水凝胶及其制备方法 |
Non-Patent Citations (4)
Title |
---|
CAO, JY; XIAO, L AND SHI, XW.: "《Injectable drug-loaded polysaccharide hybrid hydrogels for hemostasis 》", 《 RSC ADVANCES》 * |
何照范,张迪清: "《保健食品化学及其检测技术》", 31 May 1998 * |
王红梅: "《普通化学学习指导 第2版》", 30 June 2019 * |
魏文德: "《有机化工原料大全 下》", 31 August 1999 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113318276A (zh) * | 2021-03-29 | 2021-08-31 | 中山大学附属第一医院 | 一种多重交联可注射水凝胶的制备方法及其制备方法和应用 |
CN115887745A (zh) * | 2022-11-23 | 2023-04-04 | 北京航空航天大学 | 一种止血水凝胶及其制备方法和应用 |
CN115920120A (zh) * | 2022-12-01 | 2023-04-07 | 江南大学附属医院 | 促进伤口再生性愈合的水凝胶复合物及其制备方法 |
Also Published As
Publication number | Publication date |
---|---|
CN111704729B (zh) | 2022-05-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111704729B (zh) | 一种基于天然高分子且强度和成胶时间可调的水凝胶细胞支架 | |
Chan et al. | Polypeptoid polymers: Synthesis, characterization, and properties | |
Martin et al. | Enhanced stem cell retention and antioxidative protection with injectable, ROS-degradable PEG hydrogels | |
Liu et al. | Thermosensitive injectable in-situ forming carboxymethyl chitin hydrogel for three-dimensional cell culture | |
Xiao et al. | Facile strategy to construct a self-healing and biocompatible cellulose nanocomposite hydrogel via reversible acylhydrazone | |
Jin et al. | Injectable chitosan-based hydrogels for cartilage tissue engineering | |
Zamboni et al. | Labile crosslinked hyaluronic acid via urethane formation using bis (β-isocyanatoethyl) disulphide with tuneable physicochemical and immunomodulatory properties | |
Yan et al. | Injectable in situ forming poly (l-glutamic acid) hydrogels for cartilage tissue engineering | |
Jin et al. | Enzymatically-crosslinked injectable hydrogels based on biomimetic dextran–hyaluronic acid conjugates for cartilage tissue engineering | |
Yan et al. | Fabrication of injectable hydrogels based on poly (l-glutamic acid) and chitosan | |
Li et al. | Injectable conducting interpenetrating polymer network hydrogels from gelatin-graft-polyaniline and oxidized dextran with enhanced mechanical properties | |
CN109734851B (zh) | 一种温敏聚合物及其合成方法与温敏可注射水凝胶 | |
Lin et al. | Thermosensitive in situ-forming dextran–pluronic hydrogels through Michael addition | |
JP6185837B2 (ja) | ヒドロゲル前駆体調合物およびその製造プロセス | |
Li et al. | Synthesis of in-situ formable hydrogels with collagen and hyaluronan through facile Michael addition | |
Xu et al. | A fast and dual crosslinking hydrogel based on vinyl ether sodium alginate | |
Zhang et al. | Non-cytotoxic conductive carboxymethyl-chitosan/aniline pentamer hydrogels | |
CN101716346B (zh) | 一种超分子水凝胶基因载体材料及其制备方法和应用 | |
Gao et al. | Synthesis and characterization of biodegradable pH-sensitive poly (acrylic acid) hydrogels crosslinked by 2-hydroxyethyl methacrylate modified poly (L-glutamic acid) | |
Li et al. | In-situ forming biodegradable glycol chitosan-based hydrogels: synthesis, characterization, and chondrocyte culture | |
Ma et al. | In situ formation of multiple stimuli-responsive poly [(methyl vinyl ether)-alt-(maleic acid)]-based supramolecular hydrogels by inclusion complexation between cyclodextrin and azobenzene | |
Zhang et al. | Biodegradable thermo‐and pH‐responsive hydrogels for oral drug delivery | |
Li et al. | Synthesis of thiol-terminated PEG-functionalized POSS cross-linkers and fabrication of high-strength and hydrolytic degradable hybrid hydrogels in aqueous phase | |
Jing et al. | Dynamic cell-adaptable hydrogels with a moderate level of elasticity promote 3D development of encapsulated cells | |
Ying et al. | Degradable and biocompatible hydrogels bearing a hindered urea bond |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |