CN112274695A - 一种含球霰石的水凝胶支架材料的制备方法 - Google Patents
一种含球霰石的水凝胶支架材料的制备方法 Download PDFInfo
- Publication number
- CN112274695A CN112274695A CN202010998374.2A CN202010998374A CN112274695A CN 112274695 A CN112274695 A CN 112274695A CN 202010998374 A CN202010998374 A CN 202010998374A CN 112274695 A CN112274695 A CN 112274695A
- Authority
- CN
- China
- Prior art keywords
- vaterite
- solution
- hydrogel scaffold
- scaffold material
- containing hydrogel
- 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.)
- Pending
Links
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 title claims abstract description 67
- 239000000017 hydrogel Substances 0.000 title claims abstract description 50
- 239000000463 material Substances 0.000 title claims abstract description 43
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 239000000243 solution Substances 0.000 claims abstract description 60
- 108010010803 Gelatin Proteins 0.000 claims abstract description 33
- 239000008273 gelatin Substances 0.000 claims abstract description 33
- 229920000159 gelatin Polymers 0.000 claims abstract description 33
- 235000019322 gelatine Nutrition 0.000 claims abstract description 33
- 235000011852 gelatine desserts Nutrition 0.000 claims abstract description 33
- 239000005018 casein Substances 0.000 claims abstract description 17
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims abstract description 16
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims abstract description 16
- 235000021240 caseins Nutrition 0.000 claims abstract description 16
- 238000004132 cross linking Methods 0.000 claims abstract description 13
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 10
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 10
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000001110 calcium chloride Substances 0.000 claims abstract description 8
- 229910001628 calcium chloride Inorganic materials 0.000 claims abstract description 8
- 238000010382 chemical cross-linking Methods 0.000 claims abstract description 7
- 238000004108 freeze drying Methods 0.000 claims abstract description 6
- 239000011259 mixed solution Substances 0.000 claims abstract description 6
- 239000011148 porous material Substances 0.000 claims abstract description 5
- 238000002791 soaking Methods 0.000 claims description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 239000008367 deionised water Substances 0.000 claims description 13
- 229910021641 deionized water Inorganic materials 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 12
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 10
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 10
- 239000002244 precipitate Substances 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 9
- 150000002500 ions Chemical class 0.000 claims description 6
- 230000032683 aging Effects 0.000 claims description 5
- 230000035882 stress Effects 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 230000006835 compression Effects 0.000 claims description 2
- 238000007906 compression Methods 0.000 claims description 2
- 239000003519 biomedical and dental material Substances 0.000 abstract 1
- 230000007547 defect Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 9
- 102000008186 Collagen Human genes 0.000 description 3
- 108010035532 Collagen Proteins 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 229920001436 collagen Polymers 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000006185 dispersion Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000002386 leaching Methods 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 239000012266 salt solution Substances 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 230000017423 tissue regeneration Effects 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 2
- 210000000988 bone and bone Anatomy 0.000 description 2
- 230000008468 bone growth Effects 0.000 description 2
- 230000010478 bone regeneration Effects 0.000 description 2
- 229910000019 calcium carbonate Inorganic materials 0.000 description 2
- 229910001424 calcium ion Inorganic materials 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 238000012757 fluorescence staining Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 229910021532 Calcite Inorganic materials 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- -1 Salt ions Chemical class 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012669 compression test Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000006481 glucose medium Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 229920000554 ionomer Polymers 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 150000003141 primary amines Chemical class 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000001878 scanning electron micrograph Methods 0.000 description 1
- 239000012798 spherical particle Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000013077 target material Substances 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 150000003672 ureas Chemical class 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
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
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/02—Inorganic materials
- A61L27/025—Other specific inorganic materials not covered by A61L27/04 - A61L27/12
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/222—Gelatin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—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
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/52—Hydrogels or hydrocolloids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/58—Materials at least partially resorbable by the body
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
-
- 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
- A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
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)
- Inorganic Chemistry (AREA)
- Dispersion Chemistry (AREA)
- Materials For Medical Uses (AREA)
Abstract
本发明公开了一种含球霰石的新型水凝胶支架材料的制备方法。首先利用酪蛋白、Na2CO3和CaCl2合成球霰石,然后利用合成的球霰石与明胶溶液按一定比例混合,并先后通过硫酸铵溶液、N‑羟基琥珀酰亚胺(NHS)和1‑(3‑二甲氨基丙基)‑3‑乙基碳二亚胺盐酸盐(EDC)的混合溶液分别进行离子交联和化学交联,冷冻干燥后得到富含球霰石的新型水凝胶支架材料。该水凝胶支架具有良好的生物相容性和机械性能,大的孔隙率和孔径,可为组织缺损可降解支架的丰富和优化提供一种非常有潜力的可降解生物医用材料。
Description
技术领域
本发明涉及一种含球霰石的新型水凝胶支架材料的制备方法。
背景技术
天然大分子聚合物水凝胶质地柔软、富含水,可以模拟生理环境,还具有良好的生物相容性并允许营养物质在材料内转移的优点。另外,适当机械强度的水凝胶可以模拟和维持组织的生物应力环境,在促进组织再生中起着至关重要的作用。但是,水凝胶的高含水量(将近90%)总是伴随着较弱的机械强度,这在很大程度上限制了这种材料在骨骼再生中的应用。盐离子不仅影响蛋白质胶体的稳定性,而且影响其溶解度,即不同浓度的盐溶液可以增加或降低蛋白质的溶解度。基于此理论,只需将纯净的明胶凝胶浸泡在适当的盐溶液中,即可制得机械性能良好的明胶水凝胶。但是,采用盐溶液浸泡形成的离子交联明胶虽然能够提供足够的目标材料的机械强度,但是材料在体内很容易降解,不能够提供一个相对长期的细胞外基质环境。
发明内容
本发明的目的是提供一种富含球霰石的新型水凝胶支架材料的制备方法。
为了实现上述目的,本发明采用的技术方案是:
一种含球霰石的水凝胶支架材料的制备方法,其特征在于包括以下步骤:
(1)将酪蛋白溶解于Na2CO3溶液后,在搅拌下加至CaCl2溶液中,收集沉淀、洗涤、干燥获得球霰石;
(2)将球霰石分散到明胶溶液中,倒入模具中,在0~25℃下陈化,再浸泡入硫酸铵溶液进行离子交联,去离子水浸泡除去多余的硫酸铵后,再次浸泡在N-羟基琥珀酰亚胺(NHS)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)的混合溶液中进行化学交联,去离子水浸泡除去残留的NHS和EDC,冷冻干燥后得到含球霰石的水凝胶支架材料。
本发明将酪蛋白溶解于Na2CO3溶液,再加至CaCl2溶液中,酪蛋白作为软模板,得到球霰石,加料次序对于球霰石的生成非常关键,将CaCl2溶液加至溶有酪蛋白的Na2CO3溶液中不能得到球霰石。
上述步骤(1)中,酪蛋白与Na2CO3的质量比为1:1~1:5。
上述步骤(1)中,CaCl2溶液和Na2CO3溶液浓度为50~150mM,两者的物质的量之比为1:1。
上述步骤(2)中,明胶溶液的浓度为100~300 mg/mL。
上述步骤(2)中,球霰石与明胶的质量之比为1:10~1:30。
上述步骤(2)中,硫酸铵溶液的质量百分比为15~35%。
上述步骤(2)中,EDC与NHS的浓度为20~80mM。
上述步骤(2)中,制备得到的含球霰石的水凝胶支架材料的孔隙率为90-98%,孔径为120-350μm,压缩应力在0.4-2.5MPa之间,在70%的压缩应变下重复压缩20次循环没有显著的滞后现象。
天然骨组织由有机主相胶原蛋白和无机主相羟基磷灰石组成,本发明在利用胶原蛋白短肽链明胶制备的水凝胶中引入碳酸钙微球可以为骨骼再生提供钙源,能够最大化的促进骨骼生长。碳酸钙有三种无水晶体形式:球霰石,文石和方解石。其中球霰石具有多孔结构,大的比表面积,出色的生物相容性,可生物降解性等特点,球霰石在体内易逐渐溶解解离出大量的钙离子,可转化为生物羟基磷灰石,促进骨骼生长。球霰石在热力学上不稳定,采用酪蛋白作为稳定剂可合成球霰石,并且能够相对长期稳定存在。以包裹酪蛋白的球霰石为无机相,可有效地调控基于其的组织修复材料的可控降解时间,促进缺损组织修复。
本发明以明胶为有机主相,以酪蛋白为模板制备的球霰石为无机主相,通过盐溶液浸泡形成的离子交联和NHS/EDC辅助化学交联进行双交联复合的方法,可控制备一种富含球霰石的新型水凝胶支架材料,其以适宜的机械强度、可匹配新组织生成的体内降解速率,以及降解后可营养新组织形成的特点,为组织缺损可降解支架的丰富和优化提供了一种非常有潜力的可降解生物医用材料。
本发明制备方法合成工艺简易,工艺放大容易且重现性能好,采用双重协同交联赋予材料良好的机械性能;N-羟基琥珀酰亚胺(NHS)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)是两种非常重要的交联试剂。它们能够把反应体系中的羧基进行活化,催化蛋白质的羧基与伯胺发生偶联反应,交联过程中EDC和NHS不进入胶原基质中,而是转变为水溶性的脲衍生物,进一步保障了支架材料良好的生物相容性,球霰石降解可为新生骨组织提供丰富的钙离子,有助于缺损组织修复。
附图说明
图1是实施例1中球霰石的SEM图;
图2是实施例1中水凝胶支架外观图;
图3是实施例1中水凝胶支架的SEM图;
图4是实施例1中水凝胶支架压缩疲劳曲线图;
图5是实施例4中经MTT法测定的细胞增殖图;
图6是实施例4中水凝胶支架AO/EB荧光染色图。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明,均为常规方法;所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例 1富含球霰石的新型水凝胶支架材料的制备
步骤1:球霰石的制备
配制浓度为50mM的Na2CO3溶液50 mL,向其中加入0.1g的酪蛋白充分溶解。在600 r/min的搅拌下,将Na2CO3/酪蛋白溶液转移至50 mM的CaCl2溶液50mL中,搅拌20 min,离心收集沉淀,然后用去离子水洗涤3次,冷冻干燥沉淀,通过XRD检测知所获得的沉淀为球霰石,SEM检测图(图1)显示球霰石为直径1~3 μm球形颗粒。
步骤2:富含球霰石的新型水凝胶支架材料的制备
将明胶溶解于40℃水中配置成100mg/mL的溶液,以球霰石与明胶的质量比为1:10向明胶溶液中加入10mg球霰石,在600 r/min的搅拌下分散均匀。将含有球霰石的明胶溶液倒入模具中在4℃下陈化12 h,转入质量浓度为15%的硫酸铵溶液中浸泡8h进行离子交联。将离子交联后的样品用去离子水浸泡去盐,再浸泡在浓度均为30mM的NHS和EDC的混合溶液中8h进行化学交联,最后再用去离子水分多次浸泡除去残留的NHS和EDC,冷冻干燥后得到目标水凝胶支架材料。
对制备的水凝胶支架材料进行理化性质检测:图2为制备完成的水凝胶支架外观图;图3为水凝胶支架的SEM图,可见支架的孔径范围在130-300 μm;图4为水凝胶支架压缩疲劳曲线图,在70%的压缩应变下进行了20个循环后,水凝胶支架没有显示出明显的滞后现象,具有良好的弹性;对水凝胶支架材料进行压缩实验显示其压缩应力为1.6MPa。
实施例 2富含球霰石的新型水凝胶支架材料的制备
步骤1:球霰石的制备
配制浓度为60mM的Na2CO3溶液50 mL,向其中加入0.3g的酪蛋白充分溶解。在600 r/min的搅拌下,将Na2CO3/酪蛋白溶液转移至60 mM的CaCl2溶液50mL中,搅拌20 min,离心收集沉淀,然后用去离子水洗涤3次,冷冻干燥沉淀即得到球霰石,其结果与实施例1中所得的结果相似。
步骤2:富含球霰石的新型水凝胶支架材料的制备
将明胶溶解于40℃水中配置成200mg/mL的溶液,以球霰石与明胶的质量比为1:15向明胶溶液中加入15mg球霰石,在600 r/min的搅拌下分散均匀。将含有球霰石的明胶溶液倒入模具中在10℃下陈化12 h,转入质量浓度为25%的硫酸铵溶液中浸泡10h进行离子交联。将离子交联后的样品用去离子水浸泡去盐,再浸泡在浓度均为50mM的NHS和EDC的混合溶液中10h进行化学交联,最后再用去离子水分多次浸泡除去残留的NHS和EDC,冷冻干燥后得到目标水凝胶支架材料,其结果与实施例1中所得的结果相似。
实施例 3 富含球霰石的新型水凝胶支架材料的制备
步骤1:球霰石的制备
配制浓度为80mM的Na2CO3溶液50 mL,向其中加入0.4g的酪蛋白充分溶解。在600 r/min的搅拌下,将Na2CO3/酪蛋白溶液转移至80 mM的CaCl2溶液50mL中,搅拌20 min,离心收集沉淀,然后用去离子水洗涤3次,冷冻干燥沉淀即得到球霰石,其结果与实施例1中所得的结果相似。
步骤2:富含球霰石的新型水凝胶支架材料的制备
将明胶溶解于40℃水中配置成300mg/mL的溶液,以球霰石与明胶的质量比为1:20向明胶溶液中加入20mg球霰石,在600 r/min的搅拌下分散均匀。将含有球霰石的明胶溶液倒入模具中在15℃下陈化12 h,转入质量浓度为30%的硫酸铵溶液中浸泡12h进行离子交联。将离子交联后的样品用去离子水浸泡去盐,再浸泡在浓度均为70mM的NHS和EDC的混合溶液中12h进行化学交联,最后再用去离子水分多次浸泡除去残留的NHS和EDC,冷冻干燥后得到目标水凝胶支架材料,其结果与实施例1中所得的结果相似。
实施例4富含球霰石的新型水凝胶支架材料的生物相容性实验
步骤1:选取实施例1中制备的富含球霰石的水凝胶支架材料0.5g浸泡在5mL的DMEM/High Glucose 培养液(含10%的胎牛血清、100μg/ml的链霉素和100U/ml的青霉素),在37℃的生化培养箱中恒温浸泡24h±2h制备水凝胶支架材料的浸提液。
步骤2:准确称取100mg的MTT溶于20mL的PBS中,完全溶解以后用 0.22μm的滤膜过滤除菌,分装以后用锡箔纸包住置于-20℃冰箱避光保存备用。在37℃,CO2浓度为5.0%的条件下培养hBMSCs细胞。细胞传代后把hBMSCs细胞以 0.25%的胰蛋白酶(用孔径为0.22μm的膜过滤)溶液进行消化,以1×105cells/mL的细胞密度接种于96孔细胞培养板中,每孔接种100μL。将接有细胞的细胞培养板放入培养箱培养 24h后,吸去培养液,并以每5个孔为一组平行样,分别向每孔加入浸提液100μL。当培养到1d、2d和3d时,每孔分别加入浓度为5 mg/mL 的MTT溶液50μL,孵育4h后吸去上层培养液,迅速加入150μL DMSO,快速振荡5min以490nm作为检测波长,利用酶标仪测定每个孔的吸光度值。实验中使用完全培养基作为空白对照。以细胞的相对增殖率=实验组吸光度值/空白组吸光度值×100%,计算出浸提液中hBMSCs细胞的相对增殖率。细胞毒性实验检测结果(图5)表明富含球霰石的新型水凝胶支架材料的细胞毒性评价为0级。
步骤3:选取实施例1中制备的富含球霰石的水凝胶支架材料制成直径为10 mm,厚度为2 mm的圆片,用培养液浸润后置于24孔板中,在每孔中加入密度为105cells/mL的hBMSC细胞悬液100 μL,培养24h后除去上层培养液,再用PBS缓冲液清洗两遍,然后在每孔中加入200μLAO/EB荧光染色试剂,室温放置3 min后除去染色液,在荧光显微镜下观察hBMSC细胞的荧光显色状况。AO/EB荧光染色图(图6)表明所获得的水凝胶支架具有良好的生物相容性。
Claims (8)
1.一种含球霰石的水凝胶支架材料的制备方法,其特征在于包括以下步骤:
(1)将酪蛋白溶解于Na2CO3溶液后,在搅拌下加至CaCl2溶液中,收集沉淀、洗涤、干燥获得球霰石;
(2)将球霰石分散到明胶溶液中,倒入模具中,在0~25℃下陈化,再浸泡入硫酸铵溶液进行离子交联,去离子水浸泡除去多余的硫酸铵后,再次浸泡在N-羟基琥珀酰亚胺和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的混合溶液中进行化学交联,去离子水浸泡除去残留的N-羟基琥珀酰亚胺和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐,冷冻干燥后得到含球霰石的水凝胶支架材料。
2.根据权利要求1所述的含球霰石的水凝胶支架材料的制备方法,其特征在于:步骤(1)中,酪蛋白与Na2CO3的质量比为1:1~1:5。
3.根据权利要求1所述的含球霰石的水凝胶支架材料的制备方法,其特征在于:步骤(1)中,CaCl2溶液和Na2CO3溶液浓度为50~150 mM,两者的物质的量之比为1:1。
4.根据权利要求1所述的含球霰石的水凝胶支架材料的制备方法,其特征在于:步骤(2)中,明胶溶液的浓度为100~300 mg/mL。
5.根据权利要求1所述的含球霰石的水凝胶支架材料的制备方法,其特征在于:步骤(2)中,球霰石与明胶的质量之比为1:10~1:30。
6.根据权利要求1所述的含球霰石的水凝胶支架材料的制备方法,其特征在于:步骤(2)中,硫酸铵溶液的质量百分比为15~35%。
7.根据权利要求1所述的含球霰石的水凝胶支架材料的制备方法,其特征在于:步骤(2)中,N-羟基琥珀酰亚胺和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的浓度为20~80mM。
8.根据权利要求1所述的含球霰石的水凝胶支架材料的制备方法,其特征在于:步骤(2)中,制备得到的含球霰石的水凝胶支架材料的孔隙率为90-98%,孔径为120-350μm,压缩应力在0.4-2.5MPa之间,在70%的压缩应变下重复压缩20次循环没有显著的滞后现象。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010998374.2A CN112274695A (zh) | 2020-09-22 | 2020-09-22 | 一种含球霰石的水凝胶支架材料的制备方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010998374.2A CN112274695A (zh) | 2020-09-22 | 2020-09-22 | 一种含球霰石的水凝胶支架材料的制备方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN112274695A true CN112274695A (zh) | 2021-01-29 |
Family
ID=74421169
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010998374.2A Pending CN112274695A (zh) | 2020-09-22 | 2020-09-22 | 一种含球霰石的水凝胶支架材料的制备方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112274695A (zh) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114316307A (zh) * | 2021-12-31 | 2022-04-12 | 广东粤港澳大湾区国家纳米科技创新研究院 | 改性水凝胶及其制备方法和应用 |
CN114404651A (zh) * | 2022-01-25 | 2022-04-29 | 中科南京绿色制造产业创新研究院 | 一种碳酸钙复合的二肽水凝胶支架及其制备方法和应用 |
Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH0360427A (ja) * | 1989-07-28 | 1991-03-15 | Maruo Calcium Co Ltd | バテライト型炭酸カルシウム |
CN102416200A (zh) * | 2011-12-02 | 2012-04-18 | 四川大学 | 一种构建胶原基生物大分子/羟基磷灰石微球复合支架材料的制备方法 |
KR20130138948A (ko) * | 2012-06-12 | 2013-12-20 | 순천향대학교 산학협력단 | 골 재생을 위한 다공성 하이드로젤 지지체의 제조방법 |
CN104001211A (zh) * | 2014-05-04 | 2014-08-27 | 昆明理工大学 | 一种骨组织工程复合多孔支架材料及其制备方法 |
CN104692439A (zh) * | 2015-03-16 | 2015-06-10 | 浙江理工大学 | 球霰石碳酸钙微球及其制备方法 |
CN105601981A (zh) * | 2016-01-15 | 2016-05-25 | 中山大学 | 一种天然酪蛋白多孔水凝胶材料及其制备方法与应用 |
CN105727369A (zh) * | 2016-03-21 | 2016-07-06 | 中国科学院化学研究所 | 一种明胶/碳酸化羟基磷灰石骨支架的制备方法 |
US20170333190A1 (en) * | 2016-04-19 | 2017-11-23 | Warsaw Orthopedic, Inc. | Implantable composite containing carbonated hydroxyapatite |
WO2019048697A1 (en) * | 2017-09-11 | 2019-03-14 | Bone Support Ab | MACROPOROUS AND MICROPOROUS COMPOSITE CRYOGEL BIOMATERIAL FOR USE IN BONE REGENERATION |
-
2020
- 2020-09-22 CN CN202010998374.2A patent/CN112274695A/zh active Pending
Patent Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH0360427A (ja) * | 1989-07-28 | 1991-03-15 | Maruo Calcium Co Ltd | バテライト型炭酸カルシウム |
CN102416200A (zh) * | 2011-12-02 | 2012-04-18 | 四川大学 | 一种构建胶原基生物大分子/羟基磷灰石微球复合支架材料的制备方法 |
KR20130138948A (ko) * | 2012-06-12 | 2013-12-20 | 순천향대학교 산학협력단 | 골 재생을 위한 다공성 하이드로젤 지지체의 제조방법 |
CN104001211A (zh) * | 2014-05-04 | 2014-08-27 | 昆明理工大学 | 一种骨组织工程复合多孔支架材料及其制备方法 |
CN104692439A (zh) * | 2015-03-16 | 2015-06-10 | 浙江理工大学 | 球霰石碳酸钙微球及其制备方法 |
CN105601981A (zh) * | 2016-01-15 | 2016-05-25 | 中山大学 | 一种天然酪蛋白多孔水凝胶材料及其制备方法与应用 |
CN105727369A (zh) * | 2016-03-21 | 2016-07-06 | 中国科学院化学研究所 | 一种明胶/碳酸化羟基磷灰石骨支架的制备方法 |
US20170333190A1 (en) * | 2016-04-19 | 2017-11-23 | Warsaw Orthopedic, Inc. | Implantable composite containing carbonated hydroxyapatite |
WO2019048697A1 (en) * | 2017-09-11 | 2019-03-14 | Bone Support Ab | MACROPOROUS AND MICROPOROUS COMPOSITE CRYOGEL BIOMATERIAL FOR USE IN BONE REGENERATION |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114316307A (zh) * | 2021-12-31 | 2022-04-12 | 广东粤港澳大湾区国家纳米科技创新研究院 | 改性水凝胶及其制备方法和应用 |
CN114316307B (zh) * | 2021-12-31 | 2023-10-27 | 广东粤港澳大湾区国家纳米科技创新研究院 | 改性水凝胶及其制备方法和应用 |
CN114404651A (zh) * | 2022-01-25 | 2022-04-29 | 中科南京绿色制造产业创新研究院 | 一种碳酸钙复合的二肽水凝胶支架及其制备方法和应用 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP5420565B2 (ja) | セルロース誘導体からの多孔性スキャフォールドの形成方法 | |
CN100464790C (zh) | 用于骨组织工程的微支架的制备方法和应用 | |
CN111588913A (zh) | 一种自交联透明质酸及其复合胶原蛋白类的水凝胶注射剂及其应用 | |
CN112274695A (zh) | 一种含球霰石的水凝胶支架材料的制备方法 | |
CN111097068B (zh) | 一种仿生的羟基磷灰石粉体/明胶/海藻酸钠复合3d打印支架及其制备方法 | |
CN110818921B (zh) | 可快速固化的双交联水凝胶及其制备方法与应用 | |
Cai et al. | Facile synthesis of anisotropic porous chitosan/hydroxyapatite scaffolds for bone tissue engineering | |
CN111068121B (zh) | 一种木质基仿生骨支架材料的制备方法 | |
CN107638590B (zh) | 一种壳聚糖基梯度仿生复合支架材料及其构建方法 | |
Cai et al. | Fabrication and cell viability of injectable n-HA/chitosan composite microspheres for bone tissue engineering | |
CN102350009A (zh) | 复合型骨基质明胶聚乳酸多孔生物活性材料的制备方法 | |
CN114042191A (zh) | 一种细胞打印的成骨功能化支架及其制备方法和应用 | |
CN112076350B (zh) | 具有纳米-微米复合结构和高矿物质密度的仿生矿化水凝胶及其制备方法与及应用 | |
CN111249523B (zh) | 仿骨复合材料支架及其制备方法 | |
CN101791432B (zh) | 一种半乳糖化壳聚糖/聚酯类聚合物复合支架的制备方法 | |
CN112870452A (zh) | 3d打印明胶-羟基磷灰石复合水凝胶支架的制作方法 | |
CN110339403A (zh) | 球形纳米羟基磷灰石/天然高分子仿生支架及制备方法 | |
CN112028620B (zh) | 一种多孔性羟基磷灰石生物活性材料及其制备方法和应用 | |
CN101816805B (zh) | 将阿拉伯树胶用于制备骨组织修复材料的方法 | |
CN112354011B (zh) | 一种肝组织工程支架及其制备方法 | |
CN110624129B (zh) | 一种耐溶蚀的骨诱导性丝素蛋白/羟基磷灰石/氧化镁凝胶海绵及制备方法 | |
CN106512099A (zh) | 壳聚糖基仿生骨组织工程支架及其制备方法 | |
CN108904890B (zh) | 动态静电沉积复配天然材料仿生多孔微载体及制备方法 | |
CN112354016B (zh) | 一种仿生人工骨材料及其生产方法 | |
Shin et al. | Functionalizing cellulose scaffold prepared by ionic liquid with bovine serum albumin for biomedical application |
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 |