CN106620876B - 一种软骨修复水凝胶及其制备方法 - Google Patents
一种软骨修复水凝胶及其制备方法 Download PDFInfo
- Publication number
- CN106620876B CN106620876B CN201710021589.7A CN201710021589A CN106620876B CN 106620876 B CN106620876 B CN 106620876B CN 201710021589 A CN201710021589 A CN 201710021589A CN 106620876 B CN106620876 B CN 106620876B
- Authority
- CN
- China
- Prior art keywords
- repair
- plcl
- chondroitin sulfate
- cartilage
- caprolactone
- 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.)
- Expired - Fee Related
Links
- 210000000845 cartilage Anatomy 0.000 title claims abstract description 70
- 230000008439 repair process Effects 0.000 title claims abstract description 57
- 239000000017 hydrogel Substances 0.000 title claims abstract description 52
- 238000002360 preparation method Methods 0.000 title claims abstract description 28
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 claims abstract description 34
- 229920001287 Chondroitin sulfate Polymers 0.000 claims abstract description 34
- 229940059329 chondroitin sulfate Drugs 0.000 claims abstract description 34
- 239000000463 material Substances 0.000 claims abstract description 32
- 229920000428 triblock copolymer Polymers 0.000 claims abstract description 22
- 239000004698 Polyethylene Substances 0.000 claims abstract description 17
- 229920000573 polyethylene Polymers 0.000 claims abstract description 17
- -1 polyethylene Polymers 0.000 claims abstract description 11
- 239000000243 solution Substances 0.000 claims description 21
- 238000007639 printing Methods 0.000 claims description 17
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims description 12
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 12
- 239000003456 ion exchange resin Substances 0.000 claims description 12
- 229920003303 ion-exchange polymer Polymers 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 229920002567 Chondroitin Polymers 0.000 claims description 9
- 239000008367 deionised water Substances 0.000 claims description 9
- 229910021641 deionized water Inorganic materials 0.000 claims description 9
- 239000003153 chemical reaction reagent Substances 0.000 claims description 8
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 6
- JRMUNVKIHCOMHV-UHFFFAOYSA-M tetrabutylammonium bromide Chemical compound [Br-].CCCC[N+](CCCC)(CCCC)CCCC JRMUNVKIHCOMHV-UHFFFAOYSA-M 0.000 claims description 6
- VOZRXNHHFUQHIL-UHFFFAOYSA-N glycidyl methacrylate Chemical compound CC(=C)C(=O)OCC1CO1 VOZRXNHHFUQHIL-UHFFFAOYSA-N 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 238000012986 modification Methods 0.000 claims description 5
- 230000004048 modification Effects 0.000 claims description 5
- 150000001875 compounds Chemical class 0.000 claims description 4
- CTKINSOISVBQLD-UHFFFAOYSA-N Glycidol Chemical compound OCC1CO1 CTKINSOISVBQLD-UHFFFAOYSA-N 0.000 claims description 3
- 239000008055 phosphate buffer solution Substances 0.000 claims description 3
- 238000002390 rotary evaporation Methods 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 239000002202 Polyethylene glycol Substances 0.000 claims description 2
- 229920001223 polyethylene glycol Polymers 0.000 claims description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims 2
- BWZHKRSSCFRVIE-UHFFFAOYSA-N 1-n,4-n-dimethyl-2h-pyridine-1,4-diamine Chemical compound CNN1CC=C(NC)C=C1 BWZHKRSSCFRVIE-UHFFFAOYSA-N 0.000 claims 1
- QRIMLDXJAPZHJE-UHFFFAOYSA-N 2,3-dihydroxypropyl 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)OCC(O)CO QRIMLDXJAPZHJE-UHFFFAOYSA-N 0.000 claims 1
- NIXOWILDQLNWCW-UHFFFAOYSA-M Acrylate Chemical compound [O-]C(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-M 0.000 claims 1
- 239000007921 spray Substances 0.000 claims 1
- 230000006698 induction Effects 0.000 abstract description 8
- 206010007710 Cartilage injury Diseases 0.000 abstract description 4
- 230000008901 benefit Effects 0.000 abstract description 4
- 230000005847 immunogenicity Effects 0.000 abstract description 3
- 239000002994 raw material Substances 0.000 abstract description 3
- 230000000052 comparative effect Effects 0.000 description 16
- 210000004027 cell Anatomy 0.000 description 12
- 210000001519 tissue Anatomy 0.000 description 10
- 238000000034 method Methods 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 238000010146 3D printing Methods 0.000 description 7
- 238000001179 sorption measurement Methods 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 6
- 210000000963 osteoblast Anatomy 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- BAPJBEWLBFYGME-UHFFFAOYSA-N Methyl acrylate Chemical compound COC(=O)C=C BAPJBEWLBFYGME-UHFFFAOYSA-N 0.000 description 4
- 102000004067 Osteocalcin Human genes 0.000 description 4
- 108090000573 Osteocalcin Proteins 0.000 description 4
- 210000000988 bone and bone Anatomy 0.000 description 4
- 230000003013 cytotoxicity Effects 0.000 description 4
- 231100000135 cytotoxicity Toxicity 0.000 description 4
- 230000007547 defect Effects 0.000 description 4
- 230000035755 proliferation Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 210000001188 articular cartilage Anatomy 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 description 2
- 229920000437 Poly(lactide-co-caprolactone)-block-poly(ethylene glycol)-block-poly(lactide-co-caprolactone) Polymers 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 239000012620 biological material Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000003501 co-culture Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 230000008520 organization Effects 0.000 description 2
- 150000003222 pyridines Chemical class 0.000 description 2
- 238000008940 Alkaline Phosphatase assay kit Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 229920000288 Keratan sulfate Polymers 0.000 description 1
- 102000055008 Matrilin Proteins Human genes 0.000 description 1
- 108010072582 Matrilin Proteins Proteins 0.000 description 1
- 102100031475 Osteocalcin Human genes 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 230000003592 biomimetic effect Effects 0.000 description 1
- 239000002977 biomimetic material Substances 0.000 description 1
- 210000002805 bone matrix Anatomy 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- KIUKXJAPPMFGSW-MNSSHETKSA-N hyaluronan Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)C1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H](C(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-MNSSHETKSA-N 0.000 description 1
- 229940099552 hyaluronan Drugs 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000011164 ossification Effects 0.000 description 1
- 201000008968 osteosarcoma Diseases 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000009418 renovation Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
Classifications
-
- 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
- A61L27/14—Macromolecular materials
- A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
-
- 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
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
-
- 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
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Dermatology (AREA)
- Medicinal Chemistry (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Transplantation (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Dispersion Chemistry (AREA)
- Materials For Medical Uses (AREA)
Abstract
本发明属于生物医学工程材料技术领域,特别是软骨损伤修复材料制备技术领域,公开了一种软骨修复水凝胶及其制备方法。所述的软骨修复水凝胶由甲基丙烯酸化硫酸软骨素和聚丙交酯‑己内酯‑聚乙二醇‑聚丙交酯‑己内酯(PLCL‑PEG‑PLCL)三嵌段共聚物组成;按重量份数计:由甲基丙烯酸化硫酸软骨素1份、聚丙交酯‑己内酯‑聚乙二醇‑聚丙交酯‑己内酯(PLCL‑PEG‑PLCL)三嵌段共聚物1~4份组成。本发明公开的软骨修复水凝胶具有诱导修复作用、免疫原性低,原材料丰富,制备方法简单的优点。
Description
技术领域
本发明属于生物医学工程材料技术领域,特别是软骨损伤修复材料制备技术领域,具体涉及一种软骨修复水凝胶及其制备方法。
背景技术
关节软骨自我修复能力差,临床上对于关节炎和创伤导致的关节软骨损伤尚无有效修复手段。随着生物学和材料学的发展,软骨组织工程为软骨修复提供了新思路。
组织工程化软骨用于关节软骨损伤和缺损修复是目前组织工程中的研究热点。组织工程化软骨修复缺损软骨的基本原理是将自体或异体组织细胞在体外进行培养扩增后,接种到良好生物性能的支架材料上,形成细胞-支架复合体,将复合体移植至软骨缺损部位,随着支架材料的慢慢降解,组织细胞形成具有与类似软骨 功能的组织,以此达到修复缺损软骨的目的。
作为软骨组织工程的重要要素之一软骨支架材料的制备,目前存在以下问题:(1)怎么样提高支架材料的组织相容性;(2)怎样制备出一种利于细胞植入、附着、吸收、排泄等生存三维立体结构的软骨支架材料;(3)怎样提高组织材料支架的机械力学性能及表面活性;(4)材料支架的降解转归及对人体影响如何,怎样把控支架材料的降解率。所以对组织工程软骨支架的选择与制备应充分考虑到制备材料、支架类型以及制备方法的优缺点。
正常关节结构的构成,包括软骨区、软骨钙化区和骨化区等多个部分,这几个部分的结构构成是不同的,但是逐渐过渡的,它们之间没有明显的界面,因而理化性质比较稳定。然而目前组织工程软骨支架材料的制备,与人体自然软骨达不到正常过渡,容易导致组织相容性差、不易长合、应力集中、易磨损破碎等一系列问题。
软骨基质的化学成分主要为嗜碱性软骨粘蛋白,它以长链的透明质酸分子为主干,干链上以许多较短的蛋白质链连接硫酸软骨素A、C和硫酸角质素。这种羽状分支的大分子结合着大量的水,大分子又相互结合,并和胶原原纤维结合在一起形成凝胶结构。因此,水凝胶型的修复支架十分适合用于受损软骨组织的修复。但是常规方法制备的水凝胶其孔隙率,结构尺寸和微孔结构的连通性均只能控制在一定范围而不能精确在某一特定数值,其外形尺寸较单一,不能按照受损部位的情况去制作,而且常规方法所制得的水凝胶其力学强度较低。
三维打印技术(3D-printing)在生物组织工程中的研究国内外已有大量的研究报道,其中生物三维打印已在个性化医疗模型、仿生支架、药物缓释模式等方面应用,这为软骨修复材料的发展带来了新方向。生物三维打印其所打印的材料主要是以细胞、生物材料或是生长因子,利用计算机辅助软件建模后,控制打印机沉积生物材料的速度和位置,来实现仿生活性支架或是仿生材料的制造。在组织工程的应用上,生物三维打印能一步到位地根据实际所需精准地构建修复材料的内外结构,所构建的组织修复材料与正常人体组织高度相似。
发明内容
本发明的目的在于针对现有的软骨修复材料的制备精度不高,材料制备需时长,缺乏足够的力学强度、诱导修复能力和血管化能力等缺陷,提供一种具有诱导修复作用的软骨修复水凝胶及其制备方法。本发明以硫酸软骨素为原料通过甲基丙烯酸化改性制备出甲基丙烯酸化硫酸软骨素,再将其与聚丙交酯-己内酯-聚乙二醇-聚丙交酯-己内酯(PLCL-PEG-PLCL)三嵌段共聚物复合,制备出适合用于生物3D打印的打印材料,再通过生物3D打印技术制备出一种具有诱导修复作用的软骨修复水凝胶。该软骨修复水凝胶的免疫原性低,不会引起免疫排斥,材料来源丰富,具有良好的生物相容性和与正常软骨相当的力学性能,且具有诱导损伤部位修复和血管化的能力。
为实现上述目的,本发明的技术方案为:一种软骨修复水凝胶由甲基丙烯酸化硫酸软骨素和聚丙交酯-己内酯-聚乙二醇-聚丙交酯-己内酯(PLCL-PEG-PLCL)三嵌段共聚物组成;按重量份数计:由甲基丙烯酸化硫酸软骨素1份、聚丙交酯-己内酯-聚乙二醇-聚丙交酯-己内酯(PLCL-PEG-PLCL)三嵌段共聚物1~4份组成。
进一步的,所述的软骨修复水凝胶为多层圆柱状结构。
更进一步的,所述的聚丙交酯-己内酯-聚乙二醇-聚丙交酯-己内酯(PLCL-PEG-PLCL)三嵌段共聚物的分子量为8500g/mol,购自Sigma试剂公司。
为实现上述目的,本发明的另一技术方案为:一种软骨修复水凝胶的制备方法,所述的制备步骤具体如下:
(1)硫酸软骨素的甲基丙烯酸化改性:将四丁基溴化铵溶于去离子水中,配制成0.75mol/L的溶液,倒入装有离子交换树脂的层析柱中,用去离子水洗涤离子交换树脂至pH为中性;将硫酸软骨素溶于去离子水中,配制成1g/L的硫酸软骨素溶液,将硫酸软骨素溶液加入到上述层析柱中,然后收集层析柱流出的液体,置于-80℃冷冻干燥机中冷冻干燥,得硫酸软骨素-四丁基铵化合物(CS-TBA);将得到的硫酸软骨素-四丁基铵化合物(CS-TBA)溶解于二甲亚砜中,配制成30g/L的溶液,按硫酸软骨素-四丁基铵化合物(CS-TBA)、4-二甲氨基吡啶与缩水甘油基甲基丙烯酸酯的质量比为1:1:0.1,加入4-二甲氨基吡啶和缩水甘油基甲基丙烯酸酯,在50~60℃的条件下反应48h,旋转蒸发去除剩余的溶剂,得到甲基丙烯酸化硫酸软骨素;
(2)仿生打印材料的复合:将步骤(1)制备的甲基丙烯酸化硫酸软骨素与聚丙交酯-己内酯-聚乙二醇-聚丙交酯-己内酯(PLCL-PEG-PLCL)三嵌段共聚物,按质量比为1:1~4溶于PBS磷酸盐缓冲溶液中,在4℃下搅拌12 h,配制成质量浓度为10%的仿生打印材料溶液,备用;
(3)仿生水凝胶的打印:将步骤(2)配制的仿生打印材料溶液装入37℃预热的微量输出泵,打印喷头37℃预热,接收平台37℃预热;设置每层的紫外照射时间为9s,开始打印,打印完成后,用去离子水冲洗,得到软骨修复水凝胶。
进一步的,所述的离子交换树脂为50WX8型离子交换树脂,购自Sigma试剂公司;所述的四丁基溴化铵、4-二甲氨基吡啶和缩水甘油基甲基丙烯酸酯购自麦克林试剂公司。
更进一步的,所述步骤(3)仿生水凝胶的打印在RegenHU 3D 打印机上进行,所述的RegenHU 3D 打印机为光固化型3D打印机,打印喷头孔径为0.3 mm。
更进一步的,所述步骤(3)仿生水凝胶的打印模型通过辅助软件CAD或solidWord建模。
本发明的有益效果为:
(1)本发明公开的软骨修复水凝胶具有诱导修复作用、免疫原性低,原材料丰富,制备方法简单的优点。
(2)本发明以甲基丙烯酸化硫酸软骨素、聚丙交酯-己内酯-聚乙二醇-聚丙交酯-己内酯(PLCL-PEG-PLCL)三嵌段共聚物为基质,制备出3D仿生打印材料,然后利用三维打印技术进行仿生打印,不仅克服了硫酸软骨素因为其流动性能差而不能用作3D打印的问题,同时通过三维打印技术所制得的具有诱导修复作用的软骨修复水凝胶,具有与正常软骨组织相当的力学性能,且可以根据患者受损部位实际情况建模,能真正达到个性化治疗。
(3)本发明制备的软骨修复水凝胶,是采用生物三维打印技术进行仿生打印;相比于传统的组织工程软骨材料制备方法,本发明所公开的制备方法具有支架微结构精确成型,有利于生长因子或细胞的黏附。
附图说明
图1为实施例与对比例的蛋白吸附能力评价结果图;
图2为实施例和对比例与成骨细胞MG63共培养3天和7天后细胞相对增殖率图;
图3为实施例和对比例的碱性磷酸酶试验结果图;
图4为实施例和对比例的骨钙蛋白酶联吸附试验结果图。
具体实施例
下面结合实施例对本发明的技术方案做进一步的说明。
实施例1
一种软骨修复水凝胶由甲基丙烯酸化硫酸软骨素和聚丙交酯-己内酯-聚乙二醇-聚丙交酯-己内酯(PLCL-PEG-PLCL)三嵌段共聚物组成;按重量份数计:由1份甲基丙烯酸化硫酸软骨素和1份聚丙交酯-己内酯-聚乙二醇-聚丙交酯-己内酯(PLCL-PEG-PLCL)三嵌段共聚物组成。所述的软骨修复水凝胶为多层圆柱状结构,所述的聚丙交酯-己内酯-聚乙二醇-聚丙交酯-己内酯(PLCL-PEG-PLCL)三嵌段共聚物的分子量为8500g/mol,购自Sigma试剂公司。
实施例2
一种软骨修复水凝胶由甲基丙烯酸化硫酸软骨素和聚丙交酯-己内酯-聚乙二醇-聚丙交酯-己内酯(PLCL-PEG-PLCL)三嵌段共聚物组成;按重量份数计:由1份甲基丙烯酸化硫酸软骨素和2份聚丙交酯-己内酯-聚乙二醇-聚丙交酯-己内酯(PLCL-PEG-PLCL)三嵌段共聚物组成。所述的软骨修复水凝胶为多层圆柱状结构,所述的聚丙交酯-己内酯-聚乙二醇-聚丙交酯-己内酯(PLCL-PEG-PLCL)三嵌段共聚物的分子量为8500g/mol,购自Sigma试剂公司。
实施例3
一种软骨修复水凝胶由甲基丙烯酸化硫酸软骨素和聚丙交酯-己内酯-聚乙二醇-聚丙交酯-己内酯(PLCL-PEG-PLCL)三嵌段共聚物组成;按重量份数计:由1份甲基丙烯酸化硫酸软骨素和4份聚丙交酯-己内酯-聚乙二醇-聚丙交酯-己内酯(PLCL-PEG-PLCL)三嵌段共聚物组成。所述的软骨修复水凝胶为多层圆柱状结构,所述的聚丙交酯-己内酯-聚乙二醇-聚丙交酯-己内酯(PLCL-PEG-PLCL)三嵌段共聚物的分子量为8500g/mol,购自Sigma试剂公司。
实施例4
实施例1~实施例3任一例一种软骨修复水凝胶,其制备方法的具体步骤如下:
(1)硫酸软骨素的甲基丙烯酸化改性:将四丁基溴化铵溶于去离子水中,配制成0.75mol/L的溶液,倒入装有离子交换树脂的层析柱中,用去离子水洗涤离子交换树脂至pH为中性;将硫酸软骨素溶于去离子水中,配制成1g/L的硫酸软骨素溶液,将硫酸软骨素溶液加入到上述层析柱中,然后收集层析柱流出的液体,置于-80℃冷冻干燥机中冷冻干燥,得硫酸软骨素-四丁基铵化合物(CS-TBA);将得到的硫酸软骨素-四丁基铵化合物(CS-TBA)溶解于二甲亚砜中,配制成30g/L的溶液,按硫酸软骨素-四丁基铵化合物(CS-TBA)、4-二甲氨基吡啶与缩水甘油基甲基丙烯酸酯的质量比为1:1:0.1,加入4-二甲氨基吡啶和缩水甘油基甲基丙烯酸酯,在50~60℃的条件下反应48h,旋转蒸发去除剩余的溶剂,得到甲基丙烯酸化硫酸软骨素;所述的离子交换树脂为50WX8型离子交换树脂,购自Sigma试剂公司;所述的四丁基溴化铵、4-二甲氨基吡啶和缩水甘油基甲基丙烯酸酯购自麦克林试剂公司;
(2)仿生打印材料的复合:将步骤(1)制备的甲基丙烯酸化硫酸软骨素与聚丙交酯-己内酯-聚乙二醇-聚丙交酯-己内酯(PLCL-PEG-PLCL)三嵌段共聚物,按质量比为1:1~4溶于PBS磷酸盐缓冲溶液中,在4℃下搅拌12 h,配制成质量浓度为10%的仿生打印材料溶液,备用;
(3)仿生水凝胶的打印:将步骤(2)配制的仿生打印材料溶液装入37℃预热的微量输出泵,打印喷头37℃预热,接收平台37℃预热;设置每层的紫外照射时间为9s,通过辅助软件CAD或solidWord建立打印模型,在RegenHU 3D 打印机上开始进行打印,打印完成后,用去离子水冲洗,得到软骨修复水凝胶;所述的RegenHU 3D 打印机为光固化型3D打印机,打印喷头孔径为0.3 mm。
实施例5
对比例:为PVA水凝胶软骨支架(参考申请号为CN201310015460.7所公开的一种基于PVA水凝胶软骨支架的制备方法所制得)。
实验组1~3:为实施例1~3所得的软骨修复水凝胶,采用实施例4的方法制备而成。
以BSA为模型蛋白,利用BCA蛋白吸附能力测试法,评价采用实施例4的方法制备的实施例1~3与对比例的蛋白吸附能力。试验结果如图1 示。
蛋白吸附能力结果显示实施例1~3的蛋白吸附能力均较对比例要高,这与利于生长因子和营养物质在其上黏附,为细胞在其上面生长提供一个良好的微环境。而且实施例1~3得到的具有诱导修复作用的软骨修复水凝胶其蛋白吸附能力与甲基丙烯酸化硫酸软骨素在水凝胶中所占的比例呈正相关。
实施例6
对比例:为PVA水凝胶软骨支架(参考申请号为CN201310015460.7所公开的一种基于PVA水凝胶软骨支架的制备方法所制得)。
实验组1~3:为实施例1~3所得的软骨修复水凝胶,采用实施例4的方法制备而成。
阴性组:成骨细胞MG63。
将上述采用实施例4的方法制备的实施例1~3与对比例进行细胞毒性评价实验,按国标GB/T 16886.5-2003进行实验。实验结果如图2所示。
细胞毒性检测结果显示:以阴性组为参照,实施例1~3所得的软骨修复水凝胶在与成骨细胞MG63共培养3天和7天后,其对应的细胞相对增殖率均在95%以上,细胞毒性评级为0级,证明其具有良好的细胞形容性;而对比例与人成纤维细胞共培养3天和7天后,其对应细胞相对增殖率均在85%左右,细胞毒性评级为1级,无明显的细胞毒性。
此外,随着共培养时间的延长,实施例1~3所得的软骨修复水凝胶的细胞相对增殖率均有明显提高。如图2所示,实施例1~3所得的软骨修复水凝胶在共培养7天后,其细胞相对增殖率均较阴性组要高(均高于120%)。由此证明,采用本发明公开的制备方法所制得的软骨修复水凝胶具有促进成骨细胞MG63的生长,有利于新的软骨组织的生成。
实施例7
对比例:为PVA水凝胶软骨支架(参考申请号为CN201310015460.7所公开的一种基于PVA水凝胶软骨支架的制备方法所制得)。
实验组1~3:为实施例1~3所得的软骨修复水凝胶,采用实施例4的方法制备而成。
将上述采用实施例4的方法制备的实施例1~3与对比例分别与MG-63(人成骨肉瘤细胞)进行共培养7天后对其碱性磷酸酶和骨钙蛋白值进行检测,比较实验组和对比例的骨诱导能力。实验结果如图3和4所示。
碱性磷酸酶(alkaline phosphate ALP)和骨钙蛋白(osteocaltin OCN)均是分化成骨细胞的标志物,能促进骨基质的矿化。从图3和4可知,与对比例相比,实施例1~3的ALP值和骨钙蛋白含量均明显较对比例要高。由此可见,本发所制备的软骨修复水凝胶具有较高的骨诱导能力,可以促进软骨组织的诱导再生。
显然,本发明的上述实施例仅仅是为清楚地说明本发明所作的举例,而并非是对本发明的实施方式的限定;对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动,这里无需也无法对所有的实施方式予以穷举;凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明权利要求的保护范围之内。
Claims (6)
1.一种软骨修复水凝胶,其特征在于,所述的软骨修复水凝胶由甲基丙烯酸化硫酸软骨素和聚丙交酯-己内酯-聚乙二醇-聚丙交酯-己内酯(PLCL-PEG-PLCL)三嵌段共聚物组成;按重量份数计:由甲基丙烯酸化硫酸软骨素1份、聚丙交酯-己内酯-聚乙二醇-聚丙交酯-己内酯(PLCL-PEG-PLCL)三嵌段共聚物1~4份组成;
所述软骨修复水凝胶的制备方法具体步骤如下:
(1)硫酸软骨素的甲基丙烯酸化改性:将四丁基溴化铵溶于去离子水中,配制成0.75mol/L的溶液,倒入装有离子交换树脂的层析柱中,用去离子水洗涤离子交换树脂至pH为中性;将硫酸软骨素溶于去离子水中,配制成1g/L的硫酸软骨素溶液,将硫酸软骨素溶液加入到上述层析柱中,然后收集层析柱流出的液体,置于-80℃冷冻干燥机中冷冻干燥,得硫酸软骨素-四丁基铵化合物;将得到的硫酸软骨素-四丁基铵化合物溶解于二甲亚砜中,配制成30g/L的溶液,按硫酸软骨素-四丁基铵化合物、4-二甲氨基吡啶与缩水甘油基甲基丙烯酸酯的质量比为1:1:0.1,加入4-二甲氨基吡啶和缩水甘油基甲基丙烯酸酯,在50~60℃的条件下反应48h,旋转蒸发去除剩余的溶剂,得到甲基丙烯酸化硫酸软骨素;
(2)仿生打印材料的复合:将步骤(1)制备的甲基丙烯酸化硫酸软骨素与聚丙交酯-己内酯-聚乙二醇-聚丙交酯-己内酯(PLCL-PEG-PLCL)三嵌段共聚物,按质量比为1:1~4溶于PBS磷酸盐缓冲溶液中,在4℃下搅拌12h,配制成质量浓度为10%的仿生打印材料溶液,备用;
(3)仿生水凝胶的打印:将步骤(2)配制的仿生打印材料溶液装入37℃预热的微量输出泵,打印喷头37℃预热,接收平台37℃预热;设置每层的紫外照射时间为9s,开始打印,打印完成后,用去离子水冲洗,得到软骨修复水凝胶。
2.根据权利要求1所述的一种软骨修复水凝胶,其特征在于,所述的软骨修复水凝胶为多层圆柱状结构。
3.根据权利要求1所述的一种软骨修复水凝胶,其特征在于,所述的聚丙交酯-己内酯-聚乙二醇-聚丙交酯-己内酯(PLCL-PEG-PLCL)三嵌段共聚物的分子量为8500g/mol,购自Sigma试剂公司。
4.根据权利要求1所述的一种软骨修复水凝胶,其特征在于,所述的离子交换树脂为50WX8型离子交换树脂,购自Sigma试剂公司;所述的四丁基溴化铵、4-二甲氨基吡啶和缩水甘油基甲基丙烯酸酯购自麦克林试剂公司。
5.根据权利要求1所述的一种软骨修复水凝胶,其特征在于,所述步骤(3)仿生水凝胶的打印在RegenHU3D打印机上进行,所述的RegenHU3D打印机为光固化型3D打印机,打印喷头孔径为0.3mm。
6.根据权利要求1所述的一种软骨修复水凝胶,其特征在于,所述步骤(3)仿生水凝胶的打印模型通过辅助软件CAD或solidWord建模。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710021589.7A CN106620876B (zh) | 2017-01-12 | 2017-01-12 | 一种软骨修复水凝胶及其制备方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710021589.7A CN106620876B (zh) | 2017-01-12 | 2017-01-12 | 一种软骨修复水凝胶及其制备方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN106620876A CN106620876A (zh) | 2017-05-10 |
CN106620876B true CN106620876B (zh) | 2019-10-25 |
Family
ID=58844149
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201710021589.7A Expired - Fee Related CN106620876B (zh) | 2017-01-12 | 2017-01-12 | 一种软骨修复水凝胶及其制备方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN106620876B (zh) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107744602A (zh) * | 2017-09-30 | 2018-03-02 | 广东泰宝医疗器械技术研究院有限公司 | 一种可用于3d打印的生物墨水材料的制备方法 |
CN110680954A (zh) * | 2019-11-06 | 2020-01-14 | 广西医科大学 | 一种3d打印黄原胶水凝胶支架及其制备方法 |
CN114103099B (zh) * | 2021-11-24 | 2023-09-19 | 华中科技大学鄂州工业技术研究院 | 一种基于低温生物3d打印的聚己内酯-水凝胶同轴支架制备方法 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102532566A (zh) * | 2011-12-21 | 2012-07-04 | 四川大学 | 互穿网络复合水凝胶的制备方法 |
CN104645356A (zh) * | 2015-01-30 | 2015-05-27 | 复旦大学 | 一类x射线显影的热致水凝胶及其制备方法 |
CN105107019A (zh) * | 2015-09-10 | 2015-12-02 | 西南交通大学 | 一种用于关节软骨修复的红外响应高强水凝胶的制备方法 |
CN105126163A (zh) * | 2015-09-21 | 2015-12-09 | 西南交通大学 | 用于软骨修复的具有组织诱导性的水凝胶的制备方法 |
-
2017
- 2017-01-12 CN CN201710021589.7A patent/CN106620876B/zh not_active Expired - Fee Related
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102532566A (zh) * | 2011-12-21 | 2012-07-04 | 四川大学 | 互穿网络复合水凝胶的制备方法 |
CN104645356A (zh) * | 2015-01-30 | 2015-05-27 | 复旦大学 | 一类x射线显影的热致水凝胶及其制备方法 |
CN105107019A (zh) * | 2015-09-10 | 2015-12-02 | 西南交通大学 | 一种用于关节软骨修复的红外响应高强水凝胶的制备方法 |
CN105126163A (zh) * | 2015-09-21 | 2015-12-09 | 西南交通大学 | 用于软骨修复的具有组织诱导性的水凝胶的制备方法 |
Non-Patent Citations (1)
Title |
---|
Modulating rheological and degradation properties of temperature-responsive gelling systems composed of blends of PCLA–PEG–PCLA triblock copolymers and their fully hexanoyl-capped derivatives;Audrey Petit;《Acta Biomaterialia》;20121231;第8卷(第12期);第4260-4267页 * |
Also Published As
Publication number | Publication date |
---|---|
CN106620876A (zh) | 2017-05-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Osidak et al. | Collagen as bioink for bioprinting: A comprehensive review | |
Mao et al. | Fabrication of liver microtissue with liver decellularized extracellular matrix (dECM) bioink by digital light processing (DLP) bioprinting | |
Ferlin et al. | Influence of 3D printed porous architecture on mesenchymal stem cell enrichment and differentiation | |
Mellati et al. | Microengineered 3D cell‐laden thermoresponsive hydrogels for mimicking cell morphology and orientation in cartilage tissue engineering | |
Grogan et al. | Digital micromirror device projection printing system for meniscus tissue engineering | |
Wu et al. | Stiffness memory of indirectly 3D-printed elastomer nanohybrid regulates chondrogenesis and osteogenesis of human mesenchymal stem cells | |
Coburn et al. | Biomimetics of the extracellular matrix: an integrated three-dimensional fiber-hydrogel composite for cartilage tissue engineering | |
Masoumi et al. | Tri-layered elastomeric scaffolds for engineering heart valve leaflets | |
Seidlits et al. | High‐resolution patterning of hydrogels in three dimensions using direct‐write photofabrication for cell guidance | |
Raimondi et al. | Three-dimensional structural niches engineered via two-photon laser polymerization promote stem cell homing | |
Nguyen et al. | Unique biomaterial compositions direct bone marrow stem cells into specific chondrocytic phenotypes corresponding to the various zones of articular cartilage | |
Ting et al. | In vitro prefabrication of human cartilage shapes using fibrin glue and human chondrocytes | |
Kwak et al. | Formation of a keratin layer with silk fibroin-polyethylene glycol composite hydrogel fabricated by digital light processing 3D printing | |
CN106620876B (zh) | 一种软骨修复水凝胶及其制备方法 | |
Cheema et al. | Rapid fabrication of living tissue models by collagen plastic compression: understanding three-dimensional cell matrix repair in vitro | |
Bae et al. | Self-assembled extracellular macromolecular matrices and their different osteogenic potential with preosteoblasts and rat bone marrow mesenchymal stromal cells | |
CN210612586U (zh) | 一种负载软骨细胞抗炎半月板支架 | |
Chiu et al. | Engineering of oriented myocardium on three-dimensional micropatterned collagen-chitosan hydrogel | |
Hou et al. | Biodegradable and bioactive PCL–PGS core–shell fibers for tissue engineering | |
Zhang et al. | Macroporous interpenetrating network of polyethylene glycol (PEG) and gelatin for cartilage regeneration | |
Lu et al. | Macroporous methacrylated hyaluronic acid hydrogel with different pore sizes for in vitro and in vivo evaluation of vascularization | |
Hajiabbas et al. | Bioengineering in salivary gland regeneration | |
Chen et al. | Robot-assisted in situ bioprinting of gelatin methacrylate hydrogels with stem cells induces hair follicle-inclusive skin regeneration | |
Zhou et al. | Catechol functionalized ink system and thrombin-free fibrin gel for fabricating cellular constructs with mechanical support and inner micro channels | |
Xu et al. | In situ ornamenting poly (ε-caprolactone) electrospun fibers with different fiber diameters using chondrocyte-derived extracellular matrix for chondrogenesis of mesenchymal stem cells |
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 | ||
CF01 | Termination of patent right due to non-payment of annual fee | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20191025 |