CN112316211A - 一种软骨细胞外基质仿生可注射水凝胶及其制备方法 - Google Patents
一种软骨细胞外基质仿生可注射水凝胶及其制备方法 Download PDFInfo
- Publication number
- CN112316211A CN112316211A CN202011200663.XA CN202011200663A CN112316211A CN 112316211 A CN112316211 A CN 112316211A CN 202011200663 A CN202011200663 A CN 202011200663A CN 112316211 A CN112316211 A CN 112316211A
- Authority
- CN
- China
- Prior art keywords
- solution
- extracellular matrix
- cartilage
- silk fibroin
- 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
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
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3633—Extracellular matrix [ECM]
-
- 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
- A61L27/14—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
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
- A61L27/3645—Connective tissue
- A61L27/3654—Cartilage, e.g. meniscus
-
- 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
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification 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
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
-
- 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
- A61L27/54—Biologically active materials, e.g. therapeutic substances
-
- 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
-
- 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
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/06—Flowable or injectable implant compositions
-
- 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
-
- 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
- C08J2405/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Transplantation (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Dermatology (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Molecular Biology (AREA)
- Botany (AREA)
- Dispersion Chemistry (AREA)
- Organic Chemistry (AREA)
- Polymers & Plastics (AREA)
- General Chemical & Material Sciences (AREA)
- Vascular Medicine (AREA)
- Biophysics (AREA)
- Urology & Nephrology (AREA)
- Zoology (AREA)
- Materials For Medical Uses (AREA)
- Medicinal Preparation (AREA)
Abstract
本发明公开了一种软骨细胞外基质仿生可注射水凝胶及其制备方法，是通过物理作用和辣根过氧化物酶(HRP)催化交联的脱软骨细胞外基质/对羟基苯丙酸改性壳聚糖/丝素蛋白可注射水凝胶；其制备方法，是将软骨细胞外基质、对羟基苯丙酸改性壳聚糖及丝素蛋白配成溶液后在物理作用以及HRP/过氧化氢催化作用下交联形成的可注射水凝胶；该水凝胶能够在体内充填多样化的软骨缺损，并具有生物活性功能，促进软骨再生。
Description
技术领域
本发明涉及一种可注射水凝胶生物材料，具体说是脱软骨细胞外基质/改性的壳聚糖/丝素蛋白通过物理作用和辣根过氧化物酶(HRP)催化交联形成的可注射水凝胶及其制备方法。属于生物材料及生物医学工程领域。
背景技术
软骨损伤是骨科较为常见的疾患。目前对这种软骨损伤的治疗方法包括微骨折、嵌合成形术、自体软骨细胞移植和同种异体骨软骨移植，可减轻疼痛和改善关节功能，然而，这些方法形成软骨缺乏与邻近的宿主组织的良好整合度，且容易形成纤维软骨，因而影响再生软骨的质量而不能达到天然软骨组织的功能性，并可能导致进一步的并发症。因此亟待解决能用于临床的更有效的软骨组织替代物。组织工程技术可以提供替代治疗策略。具有支架材料的组织工程技术包括水凝胶和多孔支架及其与生长因子和/或干细胞复合体系具有植入物可控性、减少供体部位并发症、技术难度较小的程序及术后恢复时间较短等优势，但由于使用生长因子或细胞被食品药品监督管理局作为医疗设备和生物设备，从而产生漫长审批流程。因此，通过采用具有软骨细胞外基质成分和结构仿生的策略构建软骨组织工程支架材料，既可赋予支架生物活性和促进软骨再生，又可缩短报批时间。
发明内容
本发明针对生长因子产品审批存在审批困难以及材料与软骨不规则缺损存在整合度差问题，制备通过物理作用和辣根过氧化物酶(HRP)催化交联的脱软骨细胞外基质/壳聚糖/丝素蛋白可注射水凝胶，使其能够在体内充填多样化的软骨缺损，并具有生物活性功能，促进软骨再生。本产品采用天然软骨来源的脱软骨细胞外基质，避免使用生长因子带来的审批问题，更有利于缩短报批时间。
本发明所述软骨细胞外基质/壳聚糖/丝素蛋白可注射水凝胶，是将软骨细胞外基质、对羟基苯丙酸改性壳聚糖及丝素蛋白配成溶液后在物理作用以及HRP/过氧化氢催化作用下交联形成的可注射水凝胶。其具体制备方法包括以下步骤：
1)改性壳聚糖溶液的配制：配制0.5-1.5％g/mL壳聚糖和0.4％g/mL对羟基苯丙氨酸混合溶液，搅拌6h，加入0.726％g/mL EDC和0.346％g/mL NHS，室温搅拌48h后透析3天，冷冻干燥48h后配制成0.4-2％g/mL对羟基苯丙氨酸改性壳聚糖水溶液；
2)丝素蛋白溶液配制：称取蚕丝溶于9.3M的溴化锂溶液中配成配制20％g/mL的蚕丝溶液，置于水浴锅中60℃加热4h后，采用3500D透析袋透析48h；随后，更换20％g/mL的PEG溶液继续透析6-10h得到浓缩丝素蛋白溶液；采用10000rpm，4℃，离心20分钟后除去杂质，并通过称重法确定浓缩丝素蛋白浓度，加入适量蒸馏水配制成浓度为1-10％g/mL的溶液；
3)软骨细胞外基质制备：取新鲜猪来源新鲜软骨片加入浓度为0.01M Tris-HCl缓冲液(pH 7.5)并采用组织粉碎机粉碎成匀浆状，随后在2000-10000rpm转速下对匀浆液进行差速离心获得沉淀物；将沉淀物收集后将入浓度为1％v/v Triton X-100溶液，并于4℃下振荡12h，10000rpm离心收集并用蒸馏水重悬反复洗5次，最后一次收集沉淀；继续向沉淀中加入50U/mL Dnase I与1U/mL RNase A，并于37℃下振荡消化12h，10000rpm离心收集并用蒸馏水重悬反复洗5次，最后一次收集沉淀并冷冻于-20℃，冰冻后进行冷冻干燥，获得软骨细胞外基质冻干海绵；
4)水凝胶的制备：将0.4-2％g/mL改性壳聚糖溶液(含0.3-1.2％v/v的HRP酶溶液且浓度为10mg/mL)和0.2-10％g/mL丝素蛋白溶液(含1-4％v/v过氧化氢溶液且浓度为0.5％g/mL，含1％-3％g/mL软骨细胞外基质)等体积分别装入双管注射器的针筒内，挤压注射器使两种溶液混合形成水凝胶。
本发明与现有技术相比突出优点在于：
1)材料选取上，生物可降解的天然软骨细胞外基质、丝素蛋白和壳聚糖拥有较好的生物相容性。软骨细胞外基质保留了天然软骨组织的成分，并富含软骨再生的生长因子，可避免因直接使用生长因子而产生漫长审批流程；丝素蛋白具有良好的力学性能，可提高水凝胶力学性能；壳聚糖为线性生物高分子，具有与软骨细胞外基质中糖胺聚糖相似的结构，可补充软骨细胞外基质提取过程中损失的糖胺聚糖成分。
2)制备工艺上，利用酶交联的方法进行水凝胶制备，具有制备条件温和且无化学物质残留的特点；采用物理混合的方法将软骨细胞外基质添加到水凝胶中可最大限度保持软骨细胞外基质的生物活性。
3)产物功能上，制备的水凝胶可原位注射，可用于填充不规则软骨空洞，有利于与周围组织进行良好整合；水凝胶在成分和结构上具有与天然软骨细胞外基质相似的组成和三维结构，有利于模拟软骨细胞生存的天然环境，促进软骨组织再生。
具体实施例
实施例1：
1)配制1.5％g/mL壳聚糖和0.4％g/mL对羟基苯丙氨酸混合溶液，搅拌6h，加入0.726％g/mL EDC和0.346％g/mL NHS，室温搅拌48h后透析3天，冷冻干燥48h后配制成1％g/mL对羟基苯丙氨酸改性壳聚糖水溶液；
2)称取5g蚕丝溶于9.3M的溴化锂溶液中配成配制20％g/mL的蚕丝溶液，置于水浴锅中60℃加热4h后，采用3500D透析袋透析48h；随后，更换20％g/mL的PEG溶液继续透析10h得到浓缩丝素蛋白溶液；采用10000rpm，4℃，离心20分钟后除去杂质，并通过称重法确定浓缩丝素蛋白浓度，加入适量蒸馏水配制成浓度为5％g/mL的溶液；
3)取猪来源新鲜软骨片加入浓度为0.01M Tris-HCl缓冲液(pH 7.5)并采用组织粉碎机粉碎成匀浆状，随后在2000rpm,6000rpm和10000rpm转速下对匀浆液进行差速离心获得沉淀物；将沉淀物收集后将入浓度为1％v/v Triton X-100溶液，并于4℃下振荡12h，10000rpm离心收集并用蒸馏水重悬反复洗5次，最后一次收集沉淀；继续向沉淀中加入50U/mL Dnase I与1U/mL RNase A，并于37℃下振荡消化12h，10000rpm离心收集并用蒸馏水重悬反复洗5次，最后一次收集沉淀并冷冻于-20℃，冰冻后进行冷冻干燥，获得软骨细胞外基质冻干海绵；
4)取步骤1)中获得的5mL改性壳聚糖水溶液加入60μL浓度为10mg/mL的HRP酶，混匀后放入双管注射器A管中，然后将0.15g软骨细胞外基质溶于5mL步骤2)中获得的丝素蛋白溶液并与100μL 0.5％g/mL的过氧化氢溶液一同加入注射器B管中入，将注射器A、B管中溶液挤出，混合后即制备成水凝胶。
实施例2：
1)配制1％g/mL壳聚糖和0.4％g/mL对羟基苯丙氨酸混合溶液，搅拌6h，加入0.726％g/mL EDC和0.346％g/mL NHS，室温搅拌48h后透析3天，冷冻干燥48h后配制成1％g/mL对羟基苯丙氨酸改性壳聚糖水溶液；
2)采用实施例1中的方法提取丝素蛋白，并通过称重法确定浓缩丝素蛋白浓度，加入适量蒸馏水配制成浓度分别为0.2％，1％及5％的丝素蛋白溶液；
3)采用实施例1中的方法获得软骨细胞外基质冻干海绵；
4)取步骤1)中获得的5mL改性壳聚糖水溶液加入30μL浓度为10mg/mL的HRP酶，混匀后放入双管注射器A管中，然后将0.05g软骨细胞外基质溶于5mL步骤2)中获得的不同浓度丝素蛋白溶液并与100μL 0.5％g/mL的过氧化氢溶液一同分别加入注射器B管中入，将注射器A、B管中溶液挤出，混合后即制备成具有不同丝素蛋白含量的水凝胶。
5)通过对步骤4)获得的水凝胶进行力学测试，结果表明：随着丝素蛋白含量的增加，水凝胶的力学性能逐渐增强。
实施例3：
1)配制1％g/mL壳聚糖和0.4％g/mL对羟基苯丙氨酸混合溶液，搅拌6h，加入0.726％g/mL EDC和0.346％g/mL NHS，室温搅拌48h后透析3天，冷冻干燥48h后配制成1％g/mL对羟基苯丙氨酸改性壳聚糖水溶液；
2)采用实施例1中的方法提取丝素蛋白，并通过称重法确定浓缩丝素蛋白浓度，加入适量蒸馏水配制成浓度为5％的丝素蛋白溶液；
3)采用实施例1中的方法获得软骨细胞外基质冻干海绵；
4)取步骤1)中获得的5mL改性壳聚糖水溶液加入30μL浓度为10mg/mL的HRP酶，混匀后放入双管注射器A管中，然后分别将0.05，0.01及0.15g软骨细胞外基质溶于5mL步骤2)中获得的丝素蛋白溶液并与100μL 0.5％g/mL的过氧化氢溶液一同加入注射器B管中入，将注射器A、B管中溶液挤出，混合后即制备成具有不同软骨细胞外基质含量的水凝胶。
5)通过对步骤4)获得的水凝胶进行间充质干细胞培养，结果表明：随着软骨细胞外基质含量的增加，水凝胶更有利于间充质干细胞在材料上的黏附以及软骨分化。
Claims (2)
1.一种软骨细胞外基质仿生可注射水凝胶及其制备方法，是通过物理作用和辣根过氧化物酶(HRP)催化交联的脱软骨细胞外基质/对羟基苯丙酸改性壳聚糖/丝素蛋白可注射水凝胶。
2.所述软骨细胞外基质/壳聚糖/丝素蛋白可注射水凝胶，是将软骨细胞外基质、对羟基苯丙酸改性壳聚糖及丝素蛋白配成溶液后在物理作用以及HRP/过氧化氢催化作用下交联形成的可注射水凝胶；其具体制备方法包括以下步骤：
1)改性壳聚糖溶液的配制：配制0.5-1.5％g/mL壳聚糖和0.4％g/mL对羟基苯丙氨酸混合溶液，搅拌6h，加入0.726％g/mL EDC和0.346％g/mL NHS，室温搅拌48h后透析3天，冷冻干燥48h后配制成0.4-2％g/mL对羟基苯丙氨酸改性壳聚糖水溶液；
2)丝素蛋白溶液配制：称取蚕丝溶于9.3M的溴化锂溶液中配成配制20％g/mL的蚕丝溶液，置于水浴锅中60℃加热4h后，采用3500D透析袋透析48h；随后，更换20％g/mL的PEG溶液继续透析6-10h得到浓缩丝素蛋白溶液；采用10000rpm，4℃，离心20分钟后除去杂质，并通过称重法确定浓缩丝素蛋白浓度，加入适量蒸馏水配制成浓度为1-10％g/mL的溶液；
3)软骨细胞外基质制备：取新鲜猪来源新鲜软骨片加入浓度为0.01M Tris-HCl缓冲液(pH 7.5)并采用组织粉碎机粉碎成匀浆状，随后在2000-10000rpm转速下对匀浆液进行差速离心获得沉淀物；将沉淀物收集后将入浓度为1％v/v Triton X-100溶液，并于4℃下振荡12h，10000rpm离心收集并用蒸馏水重悬反复洗5次，最后一次收集沉淀；继续向沉淀中加入50U/mL Dnase I与1U/mL RNase A，并于37℃下振荡消化12h，10000rpm离心收集并用蒸馏水重悬反复洗5次，最后一次收集沉淀并冷冻于-20℃，冰冻后进行冷冻干燥，获得软骨细胞外基质冻干海绵；
4)水凝胶的制备：将0.4-2％g/mL改性壳聚糖溶液(含0.3-1.2％v/v的HRP酶溶液且浓度为10mg/mL)和0.2-10％g/mL丝素蛋白溶液(含1-4％v/v过氧化氢溶液且浓度为0.5％g/mL，含1％-3％g/mL软骨细胞外基质)等体积分别装入双管注射器的针筒内，挤压注射器使两种溶液混合形成水凝胶。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011200663.XA CN112316211A (zh) | 2020-10-30 | 2020-10-30 | 一种软骨细胞外基质仿生可注射水凝胶及其制备方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011200663.XA CN112316211A (zh) | 2020-10-30 | 2020-10-30 | 一种软骨细胞外基质仿生可注射水凝胶及其制备方法 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112316211A true CN112316211A (zh) | 2021-02-05 |
Family
ID=74324151
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011200663.XA Pending CN112316211A (zh) | 2020-10-30 | 2020-10-30 | 一种软骨细胞外基质仿生可注射水凝胶及其制备方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112316211A (zh) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115382021A (zh) * | 2022-09-22 | 2022-11-25 | 诺一迈尔（苏州）医学科技有限公司 | 一种复合人工软骨支架及其制备方法 |
| CN115887781A (zh) * | 2022-10-28 | 2023-04-04 | 西北大学 | 一种用于软骨损伤修复的可注射水凝胶的制备方法 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106075584A (zh) * | 2016-08-24 | 2016-11-09 | 天津市天津医院 | 可注射性软骨脱细胞外基质混合脱钙骨基质水凝胶及其制备方法 |
| CN106075597A (zh) * | 2016-06-06 | 2016-11-09 | 东华大学 | 基于膀胱脱细胞基质‑细胞‑三维纤维网络的仿生组织及其制备 |
| CN110229530A (zh) * | 2019-07-05 | 2019-09-13 | 青岛大学 | 一种双网络丝素蛋白水凝胶及其制备方法 |
| CN110464871A (zh) * | 2019-08-21 | 2019-11-19 | 南开大学 | 一种可注射水凝胶及其制备方法 |
-
2020
- 2020-10-30 CN CN202011200663.XA patent/CN112316211A/zh active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106075597A (zh) * | 2016-06-06 | 2016-11-09 | 东华大学 | 基于膀胱脱细胞基质‑细胞‑三维纤维网络的仿生组织及其制备 |
| CN106075584A (zh) * | 2016-08-24 | 2016-11-09 | 天津市天津医院 | 可注射性软骨脱细胞外基质混合脱钙骨基质水凝胶及其制备方法 |
| CN110229530A (zh) * | 2019-07-05 | 2019-09-13 | 青岛大学 | 一种双网络丝素蛋白水凝胶及其制备方法 |
| CN110464871A (zh) * | 2019-08-21 | 2019-11-19 | 南开大学 | 一种可注射水凝胶及其制备方法 |
Non-Patent Citations (9)
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115382021A (zh) * | 2022-09-22 | 2022-11-25 | 诺一迈尔（苏州）医学科技有限公司 | 一种复合人工软骨支架及其制备方法 |
| CN115382021B (zh) * | 2022-09-22 | 2024-01-05 | 诺一迈尔（苏州）医学科技有限公司 | 一种复合人工软骨支架及其制备方法 |
| CN115887781A (zh) * | 2022-10-28 | 2023-04-04 | 西北大学 | 一种用于软骨损伤修复的可注射水凝胶的制备方法 |
| CN115887781B (zh) * | 2022-10-28 | 2024-05-07 | 西北大学 | 一种用于软骨损伤修复的可注射水凝胶的制备方法 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP2307065B1 (en) | Compositions and methods for tissue filling and regeneration | |
| KR101422689B1 (ko) | 콜라겐, 히알루론산 유도체 및 포유류의 탯줄 유래 줄기세포를 포함하는 연골세포치료제 | |
| CN105169486B (zh) | 一种结合去细胞神经应用的神经修复材料 | |
| RU2529803C2 (ru) | Биологический материал, подходящий для терапии остеоартроза, повреждения связок и для лечения патологических состояний суставов | |
| KR101158776B1 (ko) | 방사선 융합 기술을 이용한 생체조직공학용 베타-글루칸 기반 지지체 및 이의 제조방법 | |
| KR101121154B1 (ko) | 지방줄기세포 또는 지방세포를 포함한 이식용 조성물 | |
| CN112316211A (zh) | 一种软骨细胞外基质仿生可注射水凝胶及其制备方法 | |
| CN111068116B (zh) | 一种注射用软骨修复温敏凝胶及其制备方法 | |
| CN108670946A (zh) | 治疗关节软骨损伤的细胞凝胶制剂及其用途和所用的保持冻存细胞活性的凝胶溶液 | |
| CN115501393B (zh) | 用于修复神经缺损的水凝胶及其制备方法和用途 | |
| CN115429937B (zh) | 一种软组织填充修复材料及其制备方法 | |
| CN111407785B (zh) | 一种用于治疗股骨头坏死的复合脱细胞基质注射液及使用方法 | |
| CN112691235A (zh) | 以物理方法为主制备人和猪脂肪源性脱细胞基质制备及应用 | |
| CN109731136B (zh) | 一种软骨诱导性基质材料的制备方法与应用 | |
| AU2004237992A1 (en) | Insoluble globin injectable implant | |
| CN113304322A (zh) | 一种用于软骨修复的角蛋白材料与细胞复合物及其制备方法 | |
| CN111714698A (zh) | 含有细胞的生物凝胶组合物、其制备方法及应用 | |
| CN111701075A (zh) | 一种软骨修复材料、软骨重建生物支架及制备方法 | |
| KR100956267B1 (ko) | 엘라스틴을 안정화제로서 함유하는 반유동성 이식용 조성물 | |
| CN109876195B (zh) | 一种可诱导快速成骨的骨填充材料的制备方法 | |
| Nussinovitch et al. | Clinical uses of alginate | |
| CN117244046A (zh) | 一种间充质干细胞组合物及其制备方法和应用 | |
| WO2022248851A1 (en) | Osteochondral device | |
| CN117752856A (zh) | 一种植骨材料及其制备方法与应用 | |
| CN117752858A (zh) | 一种引导骨生长蚕丝蛋白骨修复材料的制备方法 |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210205 |