CN106668943B - 一种装载两种细胞因子的人工骨支架及其制备方法 - Google Patents

一种装载两种细胞因子的人工骨支架及其制备方法 Download PDF

Info

Publication number
CN106668943B
CN106668943B CN201611202774.8A CN201611202774A CN106668943B CN 106668943 B CN106668943 B CN 106668943B CN 201611202774 A CN201611202774 A CN 201611202774A CN 106668943 B CN106668943 B CN 106668943B
Authority
CN
China
Prior art keywords
bone
rhbmp
rhvegf
scaffold
solution
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
Application number
CN201611202774.8A
Other languages
English (en)
Other versions
CN106668943A (zh
Inventor
许国华
鲍小刚
俞麟
朱领军
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Second Affiliated Hospital Army Medical University
Original Assignee
Second Affiliated Hospital Army Medical University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Second Affiliated Hospital Army Medical University filed Critical Second Affiliated Hospital Army Medical University
Priority to CN201611202774.8A priority Critical patent/CN106668943B/zh
Publication of CN106668943A publication Critical patent/CN106668943A/zh
Application granted granted Critical
Publication of CN106668943B publication Critical patent/CN106668943B/zh
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56Porous materials, e.g. foams or sponges
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/28Materials for coating prostheses
    • A61L27/34Macromolecular materials
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials 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
    • A61L27/3604Materials 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
    • A61L27/3608Bone, e.g. demineralised bone matrix [DBM], bone powder
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52Hydrogels or hydrocolloids
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54Biologically active materials, e.g. therapeutic substances
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L2300/00Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L2300/00Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412Tissue-regenerating or healing or proliferative agents
    • A61L2300/414Growth factors
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L2300/00Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/602Type of release, e.g. controlled, sustained, slow
    • A61L2300/604Biodegradation
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L2430/00Materials or treatment for tissue regeneration
    • A61L2430/02Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Transplantation (AREA)
  • Epidemiology (AREA)
  • Dermatology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Dispersion Chemistry (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Urology & Nephrology (AREA)
  • Zoology (AREA)
  • Botany (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Prostheses (AREA)
  • Materials For Medical Uses (AREA)

Abstract

本发明涉及医用组织工程人工骨材料技术领域,具体涉及一种装载两种细胞因子的人工骨支架及其制备方法。本发明的载两种细胞因子人工骨用于自体骨的完全骨缺损修复和重建,与传统的充填式人工骨移植方法相比,明显降低了骨折对线对位不良、软组织卡压等缺陷,并较自体骨显著促进早期骨生成。

Description

一种装载两种细胞因子的人工骨支架及其制备方法
技术领域
本发明涉及医用组织工程人工骨材料技术领域,具体涉及一种装载两种细胞因子的人工骨支架及其制备方法,该人工骨支架可完全解剖匹配骨缺损部位避免了骨折对线对位不良、软组织卡压等,显著促进了人工骨早期骨生成和后期骨整合,具有替代自体骨应用的极好潜力。
背景技术
目前利用人工骨修复大段骨缺损研究的主要突破口之一在于支架复合相关细胞因子,通过这些讯息因子的“肥料效应”诱导作用达到“间接细胞移植”,促进大段骨缺损修复和重建。将一种或多种讯息因子(如VEGFs、BMPs等)局部浓注或系统给药等传统方法具有一定的促血管生成或促成骨生成作用,但单一讯息因子往往不足以激活骨再生中的多信号通路实现最佳的骨组织再生,而且局部浓注或系统给药又存在药效时间短、副作用大等缺点[2]。随着材料学技术的发展,利用自然材料、高分子材料等包埋细胞因子调控所需讯息因子按一定浓度逐渐缓释,既能达到长期的较小有效药物刺激浓度,又能控制讯息因子扩散潜在的并发症。
针对目前临床在脊柱椎间融合,骨缺损等领域使用的美敦力“BMP-2+ACS”产品的严重副作用(骨性囊肿和异位骨化等),本实验室拟通过利用PLGA-PEG-PLGA温敏性水凝胶包埋促骨生成因子(rhBMP-2)和促血管生成因子(rhVEGF165),通过增加细胞因子的释放时间、细胞因子间的协同效应以及达到细胞因子在支架内部呈时空释放等有利因素降低BMP-2的剂量和副作用,促进大段骨缺损、椎间融合、骨不连等更好地重建和修复。
目前,尚未见装载细胞生长因子的功能化HA/PCL人工骨的相关文献报道。
发明内容
本发明的目的在于克服现有技术的不足,提供了一种装载两种细胞因子的人工骨支架及其制备方法。
本发明的主要技术方案是:
本发明的目的在于提供一种装载两种细胞因子的人工骨支架的制备方法,包括:
1)获取拟修复的自体骨CT扫描断层数据,通过三维重建技术获得骨缺损的三维模型,利用三维打印技术制备得组织工程骨;
2)将步骤1)制备的组织工程骨经消毒灭菌操作后,通过物理溶解法在负压环境下浸没于含有促骨生长因子rhBMP-2和rhVEGF-165的混合凝胶液中,于4℃静置6-10小时,保存;
3)使用时,将步骤2)浸没在混合凝胶液中的组织工程骨连同混合凝胶液一起置于37℃温育箱中静置10-15分钟,待混合凝胶液凝聚成凝胶状态后,即可。
进一步的,所述步骤1)中的消毒灭菌操作是指将组织工程骨经75%酒精浸泡12小时。
进一步的,所述步骤2)中的含有促骨生长因子rhBMP-2和rhVEGF-165的混合凝胶液,制备方法包括:
1)将PLGA-PEG-PLGA溶解于生理盐水中,制备成25wt%的水凝胶溶液40ml,将所述水凝胶溶液经过7kGy辐照消毒后置于4℃冰箱静置24h,待其成均匀溶液状态;
2)在低于25℃的无菌操作下,将水凝胶溶液与rhVEGF-165混合,配置得rhVEGF混合溶液;将水凝胶溶液与rhBMP-2混合,配置得rhBMP-2混合液;
3)将rhVEGF混合溶液与rhBMP-2混合液混匀,制得含有促骨生长因子rhBMP-2和rhVEGF-165的混合凝胶液。
进一步的,所述的温敏水凝胶PLGA-PEG-PLGA的数均分子量为1744-1500-1744,LA:GA=4:1。
进一步的,所述步骤2)中的含有促骨生长因子rhBMP-2和rhVEGF-165的混合凝胶液,rhBMP-2与rhVEGF-165的质量比为5-20:1。
进一步的,所述步骤2)中的含有促骨生长因子rhBMP-2和rhVEGF-165的混合凝胶液,优选的rhBMP-2与rhVEGF-165的质量比为10:1。
进一步的,所述的人工骨支架,其所含细胞因子浓度或量可以通过计算支架空隙体积而定量装载,细胞因子浓度或量=支架体积X孔隙率,支架体积通过移液法测得,孔隙率通过microCT测得,同时通过计算机设置调整支架孔隙率进一步调整细胞因子的装载量或浓度,直至达到满意结果。
本发明的第二目的在于提供一种通过上述制备方法所得的人工骨支架。
进一步的,所述的人工骨支架包含自然骨的皮质骨、松质骨和髓腔等部分,并与骨缺损部位匹配接合,其力学弹性模量78.33±2.82,抗压强度20.65±1.64MPa。
本发明首先制备形状可塑的解剖仿生人工骨(HA/PCL),通过计算机数字化建模筛选出合适孔径、孔隙率的人工骨,并通过万能力学机测试其抗压强度。再者,本发明通过核磁共振(1H NMR)、凝胶渗透色谱(GPC)、凝胶流变等标征和检测,优选出质量均一、变温适宜的PLGA-PEG-PLGA水凝胶,再将其与生长因子(rhBMP-2、rhVEGF-165)物理混匀可制备成可注射的原位载药凝胶,该载药凝胶在室温下呈溶胶溶液可充填多孔人工骨支架,在体温下可迅速形成固态凝胶,起到模拟红骨髓充填松质骨或骨髓腔的效果。
本发明是组织工程骨支架个体化发展的必然要求,实验通过计算机辅助技术设计3D打印的符合样本个体化解剖特点的组织工程骨支架,一方面,实验从结构上优化与控制组织工程三维支架,具有与骨缺损部位吻合的外形,有利于个性化的根据不同的骨缺损部位制备人工骨支架,同时也为一些结构不规则的结构部位的修复提供了可能。另一方面,本发明通过一种热致温敏性水凝胶包埋2种促骨生长因子(rhBMP-2、rhVEGF-165)有利于细胞因子实现支架内部时空分布和释放,模拟自然骨折修复过程中生长因子释放,为进一步组织修复研究奠定基础。
本发明的载两种细胞因子人工骨支架用于自体骨的完全骨缺损修复和重建,与传统的充填式人工骨移植方法相比,明显降低了骨折对线对位不良、软组织卡压等缺陷,并较自体骨显著促进早期骨生成。
附图说明
图1为兔胫骨CT图;其中,a为CT冠状面扫描;b为CT横断面扫描;c为CT矢状面扫描;d为CT重建骨三维结构;
图2为兔胫骨缺损的三维模型图;其中,a为自体骨缺损模型侧面观及冠状面、横断面、矢状面解剖参数;b为自体骨缺损模型底面观及解剖参数;c为自体骨缺损模上面观及解剖参数;
图3为兔胫骨缺损三维模型在FDM喷射过程中的横截面片层示意图;
图4为本发明的载两种细胞因子人工骨支架的作用示意图;
图5为兔胫骨大段完全骨缺损制备及解剖仿生人工骨植入;其中,A为根据术前电子模型量取约1.2cm胫腓骨下端截骨部位;B为完全截除自体骨留置自体骨或解剖仿生人工骨移植;C为解剖仿生人工骨;D为自体骨;E为移植及固定;F为解剖仿生人工骨移植匹配良好;
图6为兔胫骨大段完全骨缺损的术后X片扫描图;其中,图A为术后1个月,图B为术后3个月;图A和B中的a为N组;b为B组,c为VB组,d为自体骨组;
图7为术后3月的组织大体形态及CT扫描图像;其中,图a为组织大体形态,图b为CT扫描图像;图a和图b中的A为VB组,B为B组,C为N组,D为自体骨组;
图8为术后3月Van Gieson染色的组织形态观察图;其中,1为PCL片,2为新骨,3为支架,4为HA片,BM代表骨髓腔。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1载rhBMP2/rhVEGF165凝胶PLGA–PEG–PLGA的制备
1)PLGA–PEG–PLGA合成:以含有两个端羟基的PEG为引发剂、通过对丙交酯(LA)和乙交酯(GA)在辛酸亚锡催化下发生开环共聚,得到三PLGA–PEG–PLGA,并调节聚合物中亲疏水嵌段比例,合成适宜分子量的嵌段共聚物,以实现聚合物/水体系在超过凝胶浓度以后具有室温下为溶液、体温下为凝胶的热致sol-gel转变特征。(具体合成过程请参考文献(Biomacromolecules.2009Jun 8;10(6):1547-53.doi:10.1021/bm900145g.Mixing a soland a precipitate of block copolymers with different block ratios leads to aninjectable hydrogel.Yu L1,Zhang Z,Zhang H,Ding J.))
2)将PLGA-PEG-PLGA溶解于生理盐水中,制备成25wt%的水凝胶溶液40ml,该水凝胶溶液经过7kGy辐照消毒(第二军医大学辐照中心)后置于4℃冰箱静置24h待其成均匀溶液状态,在室温(低于25℃)环境下于无菌超净台中利用5ml无菌注射器抽取14ml水凝胶溶液注入含有500ug的rhVEGF165试剂瓶中,将混有水凝胶和rhVEGF的试剂瓶置于混匀器中混匀5min,配制得含VEGF浓度为36ug/ml的混合物V溶液;
3)将14ml水凝胶溶液注入含有5mg的rhBMP-2试剂瓶中配制含rhBMP-2浓度为360ug/ml的混合物B溶液;
4)取一无菌离心管(15ml)于超净台无菌操作,注射器抽取5ml V溶液和5ml B溶液,置于混匀器中混匀5min,配制得含VEGF 18ug/ml和rhBMP-2 180ug/ml的混合物VB;
5)将配置的三种凝胶溶液及不含因子的空白凝胶N溶液置于4℃冰箱待用。
实施例2解剖仿生人工骨支架的制备
以兔胫骨为例,制备解剖仿生人工骨支架:
获取兔胫骨CT(复旦大学高分子材料实验室SkyScan1176)扫描断层数据(如图1所示),利用Mimic软件的计算机三维重建技术获得骨缺损的三维模型(上海交通大学Bio-M实验室,如图2所示),计算机控制FDM喷出PCL/HA纤维作平面运动,如此层层叠加(如图3所示),制备得所需的解剖仿生人工骨支架。
实施例3装载两种细胞因子的人工骨支架的制备
1)将实施例2制备的解剖仿生人工骨支架,经75%酒精浸泡12小时左右,于超净台内严格按照无菌要求,利用1ml枪头吸取PBS液反复冲洗残留的酒精,冲洗后用无菌纱布擦干待用;
2)取6只2ml的一次性无菌真空血沉管,于超净台内严格按照无菌要求,将步骤1)备好的无菌解剖仿生人工骨支架随机装入大小合适的血沉管中,每管中3个,其中随机选取2管分别作为N组,B组和VB组;利用50ml注射器经血沉管上方的橡皮塞反复抽出血沉管内的气体使管内达到负压状态(便于水凝胶溶液充满解剖仿生人工骨内部空隙中);
3)利用3只5ml注射器分别抽取实施例1制备的空白凝胶(N溶液)、浓度为360ug/ml的rhBMP-2凝胶溶液(B溶液)和含rhVEGF165、rhBMP-2分别为18ug/ml、180ug/ml的混合凝胶溶液(VB溶液)各5ml。分别经血沉管上方的橡皮塞注入至淹没组织工程骨上部约5mm,后静置于4℃冰箱(静置6-10小时),待动物实验时将装有组织工程骨和PLGA-PEG-PLGA温敏水凝胶的3只血沉管置于37℃温育箱中静止10min左右待其凝聚成凝胶状态备用。
本实施例制备的支架,空隙体积约0.25mL,该支架可装载单一rhBMP-2(360ug/ml)以及rhVEGF-165、rhBMP-2(18ug/ml和180ug/ml)的混合液中细胞因子的总量分别为90ug以及4.5ug和45ug。
实施例4负载rhBMP2、rhVEGF165凝胶的人工骨修复兔胫骨大段骨缺损
本实施例操作利用空调调整室温到30℃,避免所制备复合体系中凝胶在较低温度时溶化。
随机选取6月龄新西兰大白兔18只(体重约3.0kg,雌雄不限),随机分为四组(N组-空白凝胶溶液、B组-BMP-2凝胶溶液、VB组-VEGF165/BMP2凝胶溶液,下同)每组6只,以及自体骨组。动物骨缺损模型制备方法及植入,如图5所示;手术操作及术后处理均为本领域的常规操作。
如图6所示,图6A为术后1月X片,红色箭头示各组示骨折处对位、对线较好,但骨折线尚明显,以B组及VB组骨支架界面修复较好;图6B为术后3月X片,红色箭头示术后3月自体骨骨折处对位、对线较好,无骨折块移位、成角畸形,骨折线模糊不明显,其中B组、VB组骨支架界面已看不见,两者表面有明显新骨增生,且VB组新骨增生较均一,B组新骨增生可见不规则裂隙。
本实施例中各组于术后1月、3月均保持了骨移植体处骨折处对位、对线较好,无骨折块移位、成角畸形等不良,说明本发明的人工骨支架结构匹配好、稳定性好;且术后1月时,该人工骨支架修复组较自体骨组明显较多的新骨生成,3月是具有相似的骨整合效果。
如图7所示,术后3月的大体形态(a)及CT扫描图像(b),各组骨端均有融合,四组均能提供良好的支撑功能,无移位、成角畸形,且存在不同程度的骨痂,其中VB组(A)可见更多的骨痂包绕组织工程骨支架(人工骨难以看清),其骨-支架界面分不出,界面处融合较紧密;B组(B)的组织工程骨周围骨痂看上去叫VB组少,其骨-支架界面尚能辨认,界面处融合紧密;N组(C)的组织工程骨周围骨痂看上去是3组中最少的,其骨-支架界面辨认清楚,界面处部分融合紧密;自体骨组(D)界面处融合较紧密。CT重建影像可见VB组(A)周围的骨痂包绕最密集,其骨-支架界面分不出;B组(B)周围骨痂不如VB组密集,但较N组组密集,其骨-支架界面辨认清楚,界面处基本完全融合;N组(C)周围无明显骨痂,其骨-支架界面辨认清楚,界面处部分融合;自体骨组(D)界面处基本完全愈合。
以上已对本发明创造的较佳实施例进行了具体说明,但本发明创造并不限于所述实施例,熟悉本领域的技术人员在不违背本发明创造精神的前提下还可做出种种的等同的变型或替换,这些等同的变型或替换均包含在本申请权利要求所限定的范围内。

Claims (4)

1.一种装载两种细胞因子的人工骨支架的制备方法,包括:
1)获取拟修复的自体骨CT扫描断层数据,通过三维重建技术获得骨缺损的三维模型,利用三维打印技术制备得组织工程骨;
2)将步骤1)制备的组织工程骨经消毒灭菌操作后,通过物理溶解法在负压环境下浸没于含有促骨生长因子rhBMP-2和rhVEGF-165的混合凝胶液中,于4℃静置6-10小时,保存;
所述步骤2)中的含有促骨生长因子rhBMP-2和rhVEGF-165的混合凝胶液,rhBMP-2与rhVEGF-165的质量比为5-20:1;
3)使用时,将步骤2)浸没在混合凝胶液中的组织工程骨连同混合凝胶液一起置于37℃温育箱中静置10-15分钟,待混合凝胶液凝聚成凝胶状态后,即可;
所述步骤2)中的含有促骨生长因子rhBMP-2和rhVEGF-165的混合凝胶液,制备方法包括:
1)将PLGA-PEG-PLGA溶解于生理盐水中,制备成25wt%的水凝胶溶液40ml,将所述水凝胶溶液经过7kGy辐照消毒后置于4℃冰箱静置24h,待其成均匀溶液状态;
2)在低于25℃的无菌操作下,将水凝胶溶液与rhVEGF-165混合,配置得rhVEGF-165混合溶液;将水凝胶溶液与rhBMP-2混合,配置得rhBMP-2混合液;
3)将rhVEGF-165混合溶液与rhBMP-2混合液混匀,制得含有促骨生长因子rhBMP-2和rhVEGF-165的混合凝胶液;
所述的PLGA-PEG-PLGA的数均分子量为1744-1500-1744,LA:GA=4:1;
所述的人工骨支架,其所含细胞因子浓度或量通过计算支架空隙体积而定量装载,细胞因子浓度或量=支架体积×孔隙率,支架体积通过移液法测得,孔隙率通过microCT测得。
2.根据权利要求1所述的制备方法,其特征在于:所述步骤2)中的消毒灭菌操作是指将组织工程骨经75%酒精浸泡12小时。
3.根据权利要求1所述的制备方法,其特征在于:所述rhBMP-2 与rhVEGF-165的质量比为10:1。
4.一种根据权利要求 1-3 任一项所述的方法制备所得的装载两种细胞因子的人工骨支架。
CN201611202774.8A 2016-12-23 2016-12-23 一种装载两种细胞因子的人工骨支架及其制备方法 Expired - Fee Related CN106668943B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201611202774.8A CN106668943B (zh) 2016-12-23 2016-12-23 一种装载两种细胞因子的人工骨支架及其制备方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201611202774.8A CN106668943B (zh) 2016-12-23 2016-12-23 一种装载两种细胞因子的人工骨支架及其制备方法

Publications (2)

Publication Number Publication Date
CN106668943A CN106668943A (zh) 2017-05-17
CN106668943B true CN106668943B (zh) 2020-07-31

Family

ID=58871263

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201611202774.8A Expired - Fee Related CN106668943B (zh) 2016-12-23 2016-12-23 一种装载两种细胞因子的人工骨支架及其制备方法

Country Status (1)

Country Link
CN (1) CN106668943B (zh)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015002707A1 (en) * 2013-05-28 2015-01-08 The Johns Hopkins University Bone regeneration using stromal vascular fraction. platelet-derived growth factor-rich hydrogel, three dimensional printed poly-epsilon-caprolactone scaffolds
CN104740686A (zh) * 2015-04-01 2015-07-01 上海交通大学医学院附属第九人民医院 一种分步式组织工程骨构建方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015002707A1 (en) * 2013-05-28 2015-01-08 The Johns Hopkins University Bone regeneration using stromal vascular fraction. platelet-derived growth factor-rich hydrogel, three dimensional printed poly-epsilon-caprolactone scaffolds
CN104740686A (zh) * 2015-04-01 2015-07-01 上海交通大学医学院附属第九人民医院 一种分步式组织工程骨构建方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PLGA-PEG-PLGA温敏水凝胶的体外释药行为;魏亚超等;《青岛科技大学学报(自然科学版)》;20140430;第35卷(第2期);摘要、第170页右栏第1段至第173页左栏第2段 *
rhBMP-2/rhVEGF-165/DPB与深低温冷冻骨成骨能力;何盛江;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20110515;摘要、文献综述 *

Also Published As

Publication number Publication date
CN106668943A (zh) 2017-05-17

Similar Documents

Publication Publication Date Title
CN103313733A (zh) 骨空隙填充剂
JP6116484B2 (ja) 脊椎固定術用の組成物および方法
Liu et al. Accelerated repair of cortical bone defects using a synthetic extracellular matrix to deliver human demineralized bone matrix
EP3530295A1 (en) Demineralized bone matrix having improved handling characteristics
Pan et al. The effects of tubular structure on biomaterial aided bone regeneration in distraction osteogenesis
Maurel et al. Bone regeneration in both small and large preclinical bone defect models using an injectable polymer‐based substitute containing hydroxyapatite and reconstituted with saline or autologous blood
Xu et al. In vitro and in vivo analysis of the effects of 3D-printed porous titanium alloy scaffold structure on osteogenic activity
CN102327643B (zh) 一种用于骨组织再生的生物支架
CN106668943B (zh) 一种装载两种细胞因子的人工骨支架及其制备方法
Zhu et al. Nano-hydroxyapatite/fibrin glue/recombinant human osteogenic protein-1 artificial bone for repair of bone defect in an animal model
WO2021050933A1 (en) Hydratable compositions comprising macroparticles and methods of making them
CN116472071A (zh) 具有高弹性的可注射磷酸钙基骨移植组合物及其制备方法
Nuntanaranont et al. Effect of chitosan scaffold on bone healing in rabbit calvarial defect.
CN110575565B (zh) 骨替代材料及其制备方法和应用
US9474828B2 (en) Self-hardening bioactive cement compositions with partially deacetylated chitin as bone graft substitutes
TWI764332B (zh) 植骨組合物
Apinun et al. Evaluation of Bone Regeneration Using Injectable Surfactant-Induced Thai Silk Fibroin/Collagen In Situ-Forming Hydrogel in Segmental Bone Defects in Rats.
JP2020031668A (ja) 医療用接着剤
WO2008094697A2 (en) Disc augmentation with hyaluronic acid
Yang et al. A mechanical-assisted post-bioprinting strategy for challenging bone defects repair
Aravind et al. In Vivo Studies of Sulphonated Polyether Ether Ketone Based Composite Bone Graft Materials.
García-Lamas et al. Enriched mesoporous bioactive glass scaffolds as bone substitutes in critical diaphyseal bone defects in rabbits
CN100493625C (zh) 生物复合人工骨及其制备方法
CN114364345A (zh) 用于脊柱融合方法的骨移植材料
Kashiwazaki et al. In vivo evaluation of a novel chitosan/HAp composite biomaterial as a carrier of rhBMP-2

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: 20200731

Termination date: 20201223